Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens

ABSTRACT

The present invention relates to compositions and methods of treating and diagnosing disorders characterized the by the presence of antigens associated with inflammatory diseases and/or cancer, and nucleotide sequences, including expressed sequence tags (ESTs), oligonucleotide probes, polypeptides, vectors and host cells expressing such antigens PRO301, PRO362 or PRO245.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the identification,isolation and recombinant production of novel DNA and novel polypeptidesthe presence of which is associated with inflammatory diseases(inflammation associated antigens) and/or cancer, and to compositionsand methods for the diagnosis and treatment of conditions characterizedby such antigens.

BACKGROUND OF THE INVENTION

[0002] The inflammatory response is complex and is mediated by a varietyof signaling molecules produced locally by mast cells, nerve endings,platelets, leucocytes and complement activation. Certain of thesesignaling molecules cause the endothelial cell lining to become moreporous and/or even to express selections which act as cell surfacemolecules which -recognize and attract leucocytes through specificcarbohydrate recognition. Stronger leucocyte binding is mediated byintegrins, which mediate leukocyte movement through the endothelium.Additional signaling molecules act as chemoattractants, causing thebound leucocytes to crawl towards the source of the attractant. Othersignaling molecules produced in the course of an inflammatory responseescape into the blood and stimulate the bone marrow to produce moreleucocytes and release them into the blood stream.

[0003] Inflammation is typically initiated by an antigen, which can bevirtually any molecule capable of initiating an immune response. Undernormal physiological conditions these are foreign molecules, butmolecules generated by the organism itself can serve as the catalyst asis known to occur in various disease states.

[0004] T-cell proliferation in a mixed lymphocyte culture or mixedlymphocyte reaction (MLR) is an established indication of the ability ofa compound to stimulate the immune system. In an inflammatory response,the responding leucocytes can be neutrophilic, eosinophilic, monocyticor lymphocytic. Histological examination of the affected tissuesprovides evidence of an immune stimulating or inhibiting response. SeeCurrent Protocols in Immunology, ed. John E. Coligan, 1994, John Wileyand Sons, Inc.

[0005] Inflammatory bowel disease (IBD) is a term used to collectivelydescribe gut disorders including both ulcerative colitis (UC) andCrohn's disease, both of which are classified as distinct disorders, butshare common features and likely share pathology. The commonality of thediagnostic criteria can make it difficult to precisely determine whichof the two disorders a patients has; however the type and location ofthe lesion in each are typically different. UC lesions arecharacteristically a superficial ulcer of the mucosa and appear in thecolon, proximal to the rectum. CD lesions are characteristicallyextensive linear fissures, and can appear anywhere in the bowel,occasionally involving the stomach, esophagus and duodenum.

[0006] Conventional treatments for IBD usually involve theadministration of antiinflammatory or immunosuppressive agents, such assulfasalazine, corticosteroids, 6-mercaptopurine/azathoprine, orcyclosporine all of which only bring partial relief to the afflictedpatient. However when antiinflammatory/immunosuppresive therapies fail,colectomies are the last line of defense. Surgery is required for about30% of CD patients within the first year after diagnosis, with thelikelihood for operative procedure increasing about 5% annuallythereafter. Unfortunately, CD also has a high rate of reoccurrence asabout 5% of patients require subsequent surgery after the initial year.UC patients further have a substantially increased risk of developingcolorectal cancer. Presumably this is due to the recurrent cycles ofinjury to the epithelium, followed by regrowth, which continuallyincreases the risk of neoplastic transformation.

[0007] A recently discovered member of the immunoglobulin superfamilyknown as Junctional Adhesion Molecule (JAM) has been identified to beselectively concentrated at intercellular junctions of endothelial andepithelial cells of different origins. Martin-Padura, I. et al., J. CellBiol. 142(1): 117-27 (1998). JAM is a typ I integral membrane proteinwith two extracellular, intrachain disulfide loops of the V-type. JAMbears substantial homology to A33 antigen (FIG. 1 or FIG. 18). Amonoclonal antibody directed to JAM was found to inhibit spontaneous andchemokine-induced monocyte transmigration through an endothelial cellmonolayer in vitro. Martin-Padura, supra

[0008] It has been recently discovered that JAM expression is increasedin the colon of CRF2-4 −/− mice with colitis. CRF 24 −/− (IL-10R subunitknockout mice) develop a spontaneous colitis mediated by lymphocytes,monocytes and neutrophils. Several of the animals also developed colonadenocarcinoma. As a result, it is foreseeable likely that the compoundsof the invention are expressed in elevated levels in or otherwiseassociated with human diseases such as inflammatory bowel disease, otherinflammatory diseases of the gut as well as colorectal carcinoma.

[0009] The compounds of the invention also bear significant homology toA33 antigen, a known colorectal cancer-associated marker. The A33antigen is expressed in more than 90% of primary or metastatic coloncancers as well as normal colon epithelium. In carcinomas originatingfrom the colonic mucosa, the A33 antigen is expressed homogeneously inmore than 95% of all cases. The A33 antigen, however, has not beendetected in a wide range of other normal tissues, i.e., its expressionappears to be organ specific. Therefore, the A33 antigen appears to playan important role in the induction of colorectal cancer.

[0010] Since colon cancer is a widespread disease, early diagnosis andtreatment is an important medical goal. Diagnosis and treatment of coloncancer can be implemented using monoclonal antibodies (mAbs) specifictherefore having fluorescent, nuclear magnetic or radioactive tags.Radioactive gene, toxins and/or drug tagged mAbs can be used fortreatment in situ with minimal patient description. mAbs can also beused to diagnose during the diagnosis and treatment of colon cancers.For example, when the serum levels of the A33 antigen are elevated in apatient, a drop of the levels after surgery would indicate the tumorresection was successful. On the other hand, a subsequent rise in serumA33 antigen levels after surgery would indicate that metastases of theoriginal tumor may have formed or that new primary tumors may haveappeared.

[0011] Such monoclonal antibodies can be used in lieu of, or inconjunction with surgery and/or other chemotherapies. For example,preclinical analysis and localization studies in patients infected withcolorectal carcinoma with a mAb to A33 are described in Welt et al., J.Clin. Oncol. 8: 1894-1906 (1990) and Welt et al., J. Clin. Oncol. 12:1561-1571 (1994), while U.S. Pat. No. 4,579,827 and U.S. Ser. No.424,991 (E.P. 199,141) are directed to the therapeutic administration ofmonoclonal antibodies, the latter of which relates to the application ofanti-A33 mAb.

SUMMARY OF THE INVENTION

[0012] The present invention further concerns compositions and methodsfor the diagnosis and treatment of inflammatory diseases in mammals,including humans. The present invention is based on the identificationof proteins (including agonist and antagonist antibodies) which eitherstimulate or inhibit the immune response in mammals. Inflammatorydiseases can be treated by suppressing the inflammatory response.Molecules that enhance an inflammatory response stimulate or potentiatethe immune response to an antigen. Molecules which stimulate aninflammatory response can be inhibited where suppression of theinflammatory response would be beneficial. Molecules which stimulate theinflammatory response can be used therapeutically where enhancement ofthe inflammatory response would be beneficial. Such stimulatorymolecules can also be inhibited where suppression of the inflammatoryresponse would be of value. Neutralizing antibodies are examples ofmolecules that inhibit molecules having immune stimulatory activity andwhich would be beneficial in the treatment of inflammatory diseases.Molecules which inhibit the inflammatory response can also be utilized(proteins directly or via the use of antibody agonists) to inhibit theinflammatory response and thus ameliorate inflammatory diseases.

[0013] Accordingly, the proteins of the invention are useful for thediagnosis and/or treatment (including prevention) of immune relateddiseases. Antibodies which bind to stimulatory proteins are useful tosuppress the inflammatory response. Antibodies which bind to inhibitoryproteins are useful to stimulate inflammatory response and the immunesystem. The proteins and antibodies of the invention are also useful toprepare medicines and medicaments for the treatment of inflammatory andimmune related diseases.

[0014] In one embodiment, the invention concerns antagonists andagonists of a PRO301, PRO362 or PRO245 polypeptide that inhibits one ormore of the functions or activities of PRO301, PRO362 or PRO245polypeptide.

[0015] In another embodiment, the invention concerns a method fordetermining the presence of a PRO301, PRO362 or PRO245 polypeptidecomprising exposing a cell suspected of containing the polypeptide to ananti-PRO301, anti-PRO362 or anti-PRO245 antibody and determining bindingof the antibody to the cell.

[0016] In yet another embodiment, the present invention relates to amethod of diagnosing an inflammatory related disease in a mammal,comprising detecting the level of expression of a gene encoding aPRO301, PRO362 or PRO245 polypeptide (a) in a test sample of tissuecells obtained from the mammal, and (b) in a control sample of knownnormal tissue cells of the same cell type, wherein a higher expressionlevel in the test sample indicates the presence of an inflammatorydisease in the mammal.

[0017] In another embodiment, the present invention relates to method ofdiagnosing an inflammatory disease in a mammal, comprising (a)contacting an anti-PRO301, anti-PRO362 or anti-PRO245 antibody with atest sample of tissue culture cells obtained from the mammal, and (b)detecting the formation of a complex between the antibody and thePRO301, PRO362 or PRO245 polypeptide. The detection may be qualitativeor quantitative, and may be performed in comparison with monitoring thecomplex formation in a control sample of known normal tissue cells ofthe same cell type. A larger quantity of complexes formed in the testsample indicates the presence of tumor in the mammal from which the testtissue cells were obtained. The antibody preferably carries a detectablelabel. Complex formation can be monitored, for example, by lightmicroscopy, flow cytometry, fluorimetry, or other techniques known inthe art. The test sample is usually obtained from an individualsuspected of having a deficiency or abnormality relating to theinflammatory response.

[0018] In another embodiment, the present invention relates to adiagnostic kit, containing an anti-PRO301, anti-PRO362 or anti-PRO245antibody and a carrier (e.g., a buffer) in suitable packaging. The kitpreferably contains instructions for using the antibody to detect thePRO301, PRO362 polypeptide.

[0019] In a further embodiment, the invention concerns an article ofmanufacture, comprising:

[0020] a container;

[0021] a label on the container; and

[0022] a composition comprising an active agent contained within thecontainer; wherein the composition is effective for stimulating orinhibiting an inflammatory response in a mammal, the label on thecontainer indicates that the composition can be used to treat aninflammatory disease, and the active agent in the composition is anagent stimulating or inhibiting the expression and/or activity of thePRO301, PRO362 or PRO245 polypeptide. In a preferred aspect, the activeagent is a PRO301, PRO362 or PRO245 polypeptide or an anti-PRO301,anti-PRO362 or anti-PRO245 antibody.

[0023] A further embodiment is a method for identifying a compoundcapable of inhibiting the expression and/or activity of a PRO301, PRO362or PRO245 polypeptide by contacting a candidate compound with a PRO301,PRO362 or PRO245 polypeptide under conditions and for time sufficient toallow these two compounds to interact. In a specific aspect, either thecandidate compound or the PRO301, PRO362 or PRO245 polypeptide isimmobilized on a solid support. In another aspect, the non-immobilizedcomponent carries a detectable label.

[0024] In yet a further aspect, the invention relates to a method oftreating an inflammatory disease, by administration of an effectivetherapeutic amount of a PRO301, PRO362 or PRO245 antagonist to a patientin need thereof for the treatment of a disease selected from:inflammatory bowel disease, systemic lupus erythematosis, rheumatoidarthritis, juvenile chronic arthritis, spondyloarthropathies, systemicsclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemic vaculitis,sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia,paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia(idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia),thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus,immune-mediated renal disease (glomerulonephritis, tubulointerstitialnephritis), demyelinating diseases of the central and peripheral nervoussystems such as multiple sclerosis, idiopathic polyneuropathy,hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C,D, E and other nonhepatotropic viruses), autoimmune chronic activehepatitis, primary biliary cirrhosis, granulomatous hepatitis, andsclerosing cholangitis, inflammatory and fibrotic lung diseases (e.g.,cystic fibrosis, eosinophilic pneumonias, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis), gluten-sensitive enteropathy,Whipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-verus host disease.

[0025] In a further embodiment, the present invention provides a methodof diagnosing tumor in a mammal, comprising detecting the level ofexpression of a gene encoding a PRO201, 362 or PRO245 polypeptide (a) ina test sample of tissue cells obtained from the mammal, and (b) in acontrol sample of known normal tissue cells of the same cell type,wherein a higher expression level in the test sample indicates thepresence of tumor in the mammal from which the test tissue cells wereobtained.

[0026] In another embodiment, the present invention provides a method ofdiagnosing tumor in a mammal, comprising (a) contacting an anti-PRO301,anti-PRO362 or anti-PRO245 antibody with a test sample of the tissuecells obtained from the mammal, and (b) detecting the formation of acomplex between the anti-PRO301, anti-PRO362 or anti-PRO245 and thePRO301, PRO362 or PRO245 polypeptide in the test sample. The detectionmay be qualitative or quantitative, and may be performed in comparisonwith monitoring the complex formation in a control sample of knownnormal tissue cells of the same cell type. A larger quantity ofcomplexes formed in the test sample indicates the presence of tumor inthe mammal from which the test tissue cells were obtained. The antibodypreferably carries a detectable label. Complex formation can bemonitored, for example, by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. Preferably, the testsample is obtained from an individual mammal suspected to haveneoplastic cell growth or proliferation (e.g., cancerous cells).

[0027] In another embodiment, the present invention provides a cancerdiagnostic kit, comprising an anti-PRO301, PRO362 or PRO245 antibody anda carrier (e.g. a buffer) in suitable packaging. The kit preferablycontains instructions for using the antibody to detect the PRO301,PRO362 or PRO245 polypeptide.

[0028] In yet another embodiment, the invention provides a method forinhibiting the growth of tumor cells comprising exposing a cell whichoverexpresses a PRO301, PRO362 or PRO245 polypeptide to an effectiveamount of an agent inhibiting the expression and/or activity of thePRO301, PRO362 or PRO245 polypeptide. The agent preferably is ananti-PRO301, anti-PRO362 or anti-PRO245 polypeptide, a small organic andinorganic peptide, phosphopeptide, antisense or ribozyme molecule, or atriple helix molecule. In a specific aspect, the agent, e.g.,anti-PRO301, anti-PRO362 or anti-PRO245 antibody induces cell death. Ina further aspect, the tumor cells are further exposed to radiationtreatment and/or a cytotoxic or chemotherapeutic agent.

[0029] In a further embodiment, the invention concerns an article ofmanufacture, comprising:

[0030] a container;

[0031] a label on the container, and

[0032] a composition comprising an active agent contained within thecontainer; wherein the composition is effective for inhibiting thegrowth of tumor cells, the label on the container indicates that thecomposition can be used for treating conditions characterized byoverexpression of a PRO301, PRO362 or PRO245 polypeptide, and the activeagent in the composition is an agent inhibiting the expression and/oractivity of the PRO301, PRO362 or PRO245 polypeptide. In a preferredaspect, the active agent is an anti-PRO301, anti-PRO362 or anti-PRO245antibody.

[0033] In a further embodiment, the invention provides an isolatednucleic acid molecule having at least about 80% sequence identity to (a)a DNA molecule encoding a PRO301 polypeptide comprising the sequence ofamino acids 28 to 258 of FIG. 2 (SEQ ID NO: 1), or (b) the complement ofthe DNA molecule of (a). The sequence identity preferably is about 85%,more preferably about 90%, most preferably about 95%. In one aspect, theisolated nucleic acid has at least about 80%, preferably at least about85%, more preferably at least about 90%, and most preferably at leastabout 95% sequence identity with a polypeptide having amino acidresidues about 28 to 235 of FIG. 2 (SEQ ID NO: 1). Preferably, thehighest degree of sequence identity occurs within the extracellulardomains (amino acids 28 to 235 of FIG. 2, SEQ ID NO: 1). In a furtherembodiment, the isolated nucleic acid molecule comprises DNA encoding aPRO301 polypeptide having amino acid residues 28 to 299 of FIG. 2 (SEQID NO: 1), or is complementary to such encoding nucleic acid sequence,and remains stably bound to it under at least moderate, and optionally,under high stringency conditions. In another aspect, the inventionprovides a nucleic acid of the full length protein of clone DNA40628,deposited with the ATCC under accession number ATCC 209432,alternatively the coding sequence of clone DNA40628, deposited underaccession number ATCC 209432.

[0034] In another embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO362 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO362polypeptide having amino acid residues 1 to 321 of FIG. 3 (SEQ ID NO:2), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions. In another aspect, the isolatednucleic acid comprises DNA encoding the PRO362 polypeptide having aminoacid residues 1 to X or FIG. 3 (SEQ ID NO: 2), where X is any amino acidresidue from 271 to 280, or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. The isolated nucleicacid sequence may comprise the cDNA insert of the DNA45416-1251 vectordeposited on Feb. 5, 1998 as ATCC 209620 which includes the nucleotidesequence encoding PRO362.

[0035] In yet another embodiment, the invention provides isolatednucleic acid molecules that hybridize to the complement of the nucleicacid molecules encoding the PRO301, PRO362 or PRO245 polypeptides. Thenucleic acid preferably is DNA, and hybridization occurs under stringentconditions. Such nucleic acid molecules can act as antisense moleculesof the inflammation associated antigens identified herein, which, inturn, can find use in the modulation of the inflammation associatedantigens, or as antisense primers in amplification reactions.Furthermore, such sequences can be used as part of ribozyme and/ortriple helix sequence which, in turn, may be used in regulation of theinflammation associated antigens.

[0036] In yet another embodiment, the invention provides a vectorcomprising DNA encoding PRO301 or a PRO362 polypeptide. A host cellcomprising such a vector is also provided. By way of example, the hostcells may be CHO cells, E. coli, or yeast. A process for producingPRO301 or PRO362 polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of PRO301or PRO362 and recovering the same from the cell culture.

[0037] In yet another embodiment, the invention provides isolated PRO301polypeptide. In particular, the invention provides isolated nativesequence PRO301 polypeptide, which in one embodiment, includes an aminoacid sequence comprising the extracellular domain residues 28 to 235 ofFIG. 2 (SEQ ID NO: 1). Native PRO301 polypeptides with or without thenative signal sequence (amino acids 1 to 27 in FIG. 2 (SEQ ID NO: 1),and with or without the initiating methionine are specifically included.Additionally, the sequences of the invention may also comprise thetransmembrane domain (residues 236 to 258 in FIG. 2)(SEQ ID NO: 1)and/or the intracellular domain (residue 259 to 299 in FIG. 2)(SEQ IDNO: 1). Alternatively, the invention provides a PRO301 polypeptideencoded by the nucleic acid deposited under accession number ATCC209432.

[0038] In yet another embodiment, the invention provides isolated PRO362polypeptide. In particular, the invention provides isolated nativesequence PRO362, which in one aspect, includes an amino acid sequencecomprising residues 1 to 321 of FIG. 3 (SEQ ID NO: 2). An additionalembodiment of the present invention is directed to an isolatedextracellular domain of a PRO362 polypeptide comprising amino acids 1 toX of the FIG. 2 (SEQ ID NO: 2), wherein X is any amino acid residue271-280. Optionally, the PRO362 polypeptide is obtained or is obtainableby expressing the polypeptide encoded by the cDNA insert of theDNA45416-1251 vector deposited on Feb. 5, 1998 as ATCC Deposit No.209620.

[0039] In yet another embodiment, the invention provides chimericmolecules comprising a PRO301 or PRO362 polypeptide fused to aheterologous polypeptide or amino acid sequence. An example of such achimeric molecule comprises a PRO301 or PRO362 polypeptide fused to anepitope tag sequence or a Fc region of an immunoglobulin.

[0040] In yet another embodiment, the invention provides an expressedsequence tag (EST) comprising the nucleotide sequences identified as:DNA35936 (SEQ ID NO: 3) in FIG. 4A, consen01 (SEQ ID NO: 4) in FIG. 4Band consen02 (DNA42257)(SEQ ID NO: 5).

[0041] In another embodiment, the present invention provides an isolatedantibody which binds a PRO301 or PRO362 polypeptide. In one aspect, theantibody mimics the activity of a PRO301 or PRO362 polypeptide (anagonist antibody) or conversely the antibody inhibits or neutralizes theactivity of a PRO301 or PRO362 polypeptide (antagonist antibody). Inanother aspect, the antibody is a monoclonal antibody, which preferablycontains nonhuman complementarity determining region (CDR) residues andhuman framework region (FR) residues. The antibody may be labeled and/orimmobilized on a solid support. In a further aspect, the antibody isaffinity matured, an antibody fragment, a single-chain antibody, or ananti-idiotypic antibody.

[0042] In another embodiment, the invention provides a compositioncontaining a PRO301 or PRO362 polypeptide or an agonist or antagonistantibody in admixture with a carrier or excipient. In one aspect, thecomposition contains a therapeutically affective amount of the peptideor antibody. In another aspect, when the composition contains aninflammation stimulating molecule, the composition is useful for: (a)increasing infiltration of inflammatory cells into a tissue of a mammalin need thereof, (b) stimulating or enhancing an immune response in amammal in need thereof, or (c) increasing the proliferation ofT-lymphocytes in a mammal in need thereof in response to an antigen. Ina further aspect, when the composition contains an inflammatoryinhibiting molecule, the composition is useful for: (a) decreasinginfiltration of inflammatory cells into a tissue of a mammal in needthereof, (b) inhibiting or reducing an inflammatory response in a mammalin need thereof, or (c) decreasing the proliferation of T-lymphocytes ina mammal in need thereof in response to an antigen. In another aspect,the composition contains a further active ingredient, which may, forexample, be a further antibody or a cytotoxic or chemotherapeutic agent.Preferably, the composition is sterile.

[0043] In a further embodiment, the invention concerns nucleic acidencoding an anti-PRO301 and anti-PRO362 antibody, and vectors andrecombinant host cells comprising such nucleic acid. In a still furtherembodiment, the invention concerns a method for producing such anantibody by culturing a host cell transformed with nucleic acid encodingthe antibody under conditions such that the antibody is expressed, andrecovering the antibody from the cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 show a comparison between the polypeptides encoded by A33antigen (SEQ ID NO: 6), DNA40628 (SEQ ID NO: 1), DNA45416 (SEQ ID NO:2), DNA35638 (SEQ ID NO: 9) and JAM (SEQ ID NO: 10).

[0045]FIG. 2 shows the derived amino acid sequence (SEQ ID NO: 1) of anative sequence PRO301 polypeptide. This polypeptide is 299 amino acidslong, having signal sequence at residue 1 to 27, an extracellular domainat residue 28 to about 235, Ig superfamily homology at residue 94 to235, a potential transmembrane domain at residue 236 to about 258, andan intracellular domain at about residue 259 to 299.

[0046]FIG. 3 shows the amino acid sequence (SEQ ID NO: 2) derived fromnucleotides 119-1081 of the nucleotide sequence shown in FIG. 6A and 6B(DNA45416, SEQ ID NO: 7). Also shown in FIG. 3 as underlines are thelocations of a glycosoaminoglycan site and a transmembrane domain.

[0047]FIG. 4A shows the consensus assembly DNA35936 (SEQ ID NO: 3), andFIG. 4B shows consen01 (SEQ ID NO: 4) which were both used in theisolation of DNA40628 (SEQ ID NO: 11). FIG. 4C shows consen02 (DNA42257)(SEQ ID NO: 5) which was used in the isolation of DNA45416 (SEQ ID NO:7).

[0048]FIG. 5 shows the nucleotide sequence of a native sequence DNA40628cDNA (SEQ ID NO: 11), which is a native sequence PRO301 cDNA alsodesignated as “UNQ264” and/or “DNA40628-1216”.

[0049]FIGS. 6A & B show a nucleotide sequence DNA45416 (SEQ ID NO: 7)which is a native sequence PRO362 cDNA also designated as “UNQ317”and/or “DNA45416-1251”. Also presented is the initiator methionine andthe protein translation for a full-length PRO362 polypeptide (SEQ ID NO:2).

[0050]FIG. 7 shows the nucleotide sequence (SEQ ID NO: 8) of a nativesequence PRO245 cDNA, wherein the nucleotide sequence is designated as“UNQ219” and/or “DNA35638”.

[0051]FIG. 8 shows the oligonucleotide sequences OLI2162 (35936.f1)(SEQID NO: 12), OLI2163 (35936.p1)(SEQ ID NO: 13), OLI2164 (35936.f2)(SEQ IDNO: 14), OLI2165 (35936.r1) (SEQ ID NO: 15) OLI2166 (35936.f3)(SEQ IDNO: 16), OLI2167 (35936.r2)(SEQ ID NO: 17) which were used in theisolation of DNA40628.

[0052]FIG. 9 shows a double stranded representation of the DNA42257(consen02) (SEQ ID NO: 5) along with the locations of fiveoligonucleotide primers, showed in underline, all used in the isolationof DNA45416 (SEQ ID NO: 7). The oligonucleotides depicted are: 42257.fI(SEQ ID NO: 18), 42257.f2 (SEQ ID NO: 19), 42257.r1 (SEQ ID NO: 20),42257.r2 (SEQ ID NO: 21) and 42257.p1 (SEQ ID NO: 22).

[0053]FIG. 10 describes the Blast score, match and percent homologyalignment between 2 overlapping fragments of DNA40628 and A33_HUMAN, ahuman A33 antigen precursor. FIG. 10A compares the coded residuesbeginning at nucleotide position 121 to 816 of DNA40628 (SEQ ID NO: 23)with the coded residues beginning at nucleotides 17 to 284 of A33_HUMAN(SEQ ID NO: 24); FIG. 10B compares the coded residues beginning atnucleotides 112 to 810 (SEQ ID NO: 25) with the coded residues beginningat nucleotides 12 to 284 (SEQ ID NO: 26), respectively.

[0054]FIG. 11 shows the derived amino acid sequence of a native sequencePRO245 polypeptide (SEQ ID NO: 9) encoded by the nucleotide sequence ofFIG. 7 (DNA35638, SEQ ID NO: 8).

[0055]FIG. 12 indicates a 25.3% identity between the amino acid sequenceencoded by DNA40628 (SEQ ID NO: 1) and A33 antigen (SEQ ID NO: 6).

[0056]FIG. 13 indicates a 20.8% identity between the amino acid sequenceencoded by DNA45416 (SEQ ID NO: 2) and A33 antigen (SEQ ID NO: 6).

[0057]FIG. 14 indicates a 24.3% identity between the amino acid sequenceencoded by DNA35638 (SEQ ID NO: 9) and A33 antigen (SEQ ID NO: 6).

[0058]FIG. 15 indicates a 67.6% identity between the amino acid sequenceencoded by DNA40628 (SEQ ID NO: 1) and JAM (SEQ ID NO: 10).

[0059]FIG. 16 indicates a 23.3% identity between the amino acid sequenceencoded by DNA45416 (SEQ ID NO: 2) and JAM (SEQ ID NO: 10).

[0060]FIG. 17 indicates a 34.2% identity between the amino acid sequenceencoded by DNA35638 (SEQ ID NO: 29) and JAM (SEQ ID NO: 10).

[0061]FIG. 18 indicates a 26% identity between the amino acid sequenceencoded by A33 antigen (SEQ ID NO: 6) and JAM (SEQ ID NO: 10).

[0062]FIG. 19 shows the results of the dot blot hybridization proceduredescribed in Example 8

[0063]FIG. 20 shows the results of the Taqman MRNA expression assaydescribed in Example 9

[0064]FIG. 21 shows the binding of protein encoded by DNA40628 to humanneutrophils as described in Example 7.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

[0065] The terms “PRO301”, “PRO362, “PRO245”, or “PRO301 polypeptide,”“PRO362 polypeptide,” “PRO245 polypeptide” and “cancer associatedantigen” when used herein encompass native sequence PRO301, PRO362 orPRO245, respectively and variants thereof (which are further definedherein). The PRO301, PRO362 or PRO245 may be isolated from a variety ofsources, such as from human tissue types or from another source, orprepared by recombinant or synthetic methods.

[0066] The terms “inflammatory disease” means a disease in which acomponent of the immune system of a mammal causes, mediates or otherwisecontributes to an inflammatory response contributing to the morbidity inthe mammal. Also included are diseases in which stimulation orintervention of the inflammatory response has an ameliorative effect onprogression of the disease. Included within this term areimmune-mediated inflammatory diseases.

[0067] The term “T-cell mediated” disease means a disease in which Tcells directly or indirectly mediate or otherwise contribute to themorbidity in a mammal. The T cell mediated disease by be associated withcell mediated effects, lymphokine mediated effects, etc. and eveneffects associated with B cells if the B cells are stimulated, forexample; by the lymphokines secreted by T cells.

[0068] Examples of immune-related and inflammatory diseases, some ofwhich are T cell mediated, which can be treated according to theinvention include: inflammatory bowel disease, systemic lupuserythematosis, rheumatoid arthritis, juvenile chronic arthritis,spondyloarthropathies, systemic sclerosis (scleroderma), idiopathicinflammatory myopathies (dermatomyositis, polymyositis), Sjögren'ssyndrome, systemic vaculitis, sarcoidosis, autoimmune hemolytic anemia(immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other nonhepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory and fibrotic lungdiseases (e.g., cystic fibrosis), gluten-sensitive enteropathy,Whipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-verus host disease. “Tumor”, as used herein, refersto all neoplastic cell growth and proliferation whether malignant orbenigh, and all pre-cancerous cells and tissues.

[0069] The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typcially characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include breast cancer, prostatecancer, colon cancer, squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, colorectal cancer, endometrial carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

[0070] “Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In treatment of an immune related disease,a therapeutic agent may directly decrease or increase the magnitude ofresponse of a component of the immune response, or render the diseasemore susceptible to treatment by other therapeutic agents, e.g.,antibiotics, antifungals, anti-inflammatory agents, chemotherapeutics,etc.

[0071] The “pathology” of an immune related disease includes allphenomena that compromise the well-being of the patient. This includes,without limitation, abnormal or uncontrollable cell growth(neutrophilic, eosinophilic, monocytic, lymphocytic cells), antibodyproduction, auto-antibody production, complement production,interference with the normal functioning of neighboring cells, releaseof cytokines or other secretory products at abnormal levels, suppressionor aggravation of any inflammatory or immunological response,infiltration of inflammatory cells (neutrophilic, eosinophilic,monocytic, lymphocytic) into cellular spaces, etc.

[0072] The terms “mammal” as used herein refers to any mammal classifiedas a mammal, including humans, domestic and farm animals, and zoo,sports or pet animals such horses, pigs, cattle, dogs, cats and ferrets,etc. In a preferred embodiment of the invention, the mammal is a human.

[0073] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0074] The term “cytotoxic agent” as used herein refers to a substancethat inhibits or prevents the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g. I¹³¹, I¹²⁵, y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, andtoxins such a enzymatically active toxins of bacterial, fungal, plant oranimal origin, or fragments thereof.

[0075] A “chemotherapeutic agent” is a compound useful in the treatmentof cancer. Examples of chemotherapeutic agents include adriamycin,doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside (“Ara-C”),cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g. paclitaxel(Taxol®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel(Taxotere®, Rhone-Poulenc Roher, Antony, France), toxotere,methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide,ifosfamide, mitomycin C, mitoxantrone, vincristine (Loucristine),vinorelbine, carboplatin, teniposide, daunomycin, carminomycin,aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Pat. No.4,675,187), melphalan and other related nitrogen mustards. Also includedin this definition are hormonal agents that act to regulate or inhibithormonal action on tumors such as tamoxifen and onapristone.

[0076] A “growth inhibitory agent” when used herein refers to a compoundor composition which inhibits growth of a cell, especially cancers cellsexpressing or overexpressing any of the genes identified herein, eitherin vitro or in vivo. Thus, the growth inhibitory agent is one whichsignificantly reduces the percentage of cells expressing oroverexpressing such genes in S phase. Examples of growth inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vinca alkaloids(vincristine and vinblastine), taxol, and topo II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogens, and antineoplastic drugs” by Murakami et al. (W B Saunders,Philadelphia, 1995), especially page 13.

[0077] The term “cytokine” is a generic term for proteins released byone cell population which act on another cell as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone, parathyroid hormone, thyroxine,insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH), hepatic growth factor, fibroblast growthfactor, prolactin, placental lactogen, tumor necrosis factor-α and -β,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,thrombopoietin (TPO), nerve growth factors such as NGF-β,platelet-growth factor, transforming growth factors (TGFs) such as TGF-αand TGF-β, insulin-like growth factor-I and -II, erythropoietin (EPO),osteoinductive factors, interferons such as interferon- α, -β, and -γ,colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF),granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF),interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12, a tumor necrosis factor such as THF-α orTNF-β, and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

[0078] “Therapeutically effective amount” is the amount of activePRO301, PRO362 or PRO245 antagonist or agonist which is required toachieve a measureable inhibition or stimulation, as the case may be, ofthe inflammatory response.

[0079] A “native sequence PRO301, PRO362 or PRO245”, comprises apolypeptide having the same amino acid sequence as a PRO301, PRO362 orPRO245, respectively, derived from nature. Such native sequence PRO301,PRO362 or PRO245 can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native sequence PRO301”,“native sequence PRO362” or “native sequence PRO245” specificallyencompasses naturally-occurring truncated or secreted forms of PRO301,PRO362 or PRO245, respectively (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of PRO301, PRO362 or PRO245,respectively.

[0080] In one embodiment, the native sequence PRO301 is a mature orfull-length native sequence PRO301 comprising amino acids 1 to 299 ofFIG. 2 (SEQ ID NO: 1), with or without the N-terminal signal sequence,with or without the initiating methionine at position 1, with or withoutthe potential transmembrane domain at position 236 to about 258, andwith or without the intracellular domain at about position 259 to 299.

[0081] In another embodiment, the native sequence PRO362 polypeptide isan extracellular domain of the full-length PRO362 protein comprisingamino acids 1 to X of the amino acid sequence shown in FIG. 3 (SEQ IDNO: 2) where X is any amino acid residue 271-280. Optionally, the PRO362polypeptide is obtained or obtainable by expressing the polypeptideencoded by the cDNA insert of the vector DNA45416-1251 deposited on Feb.5, 1998 as ATCC Deposit No.: 209620.

[0082] In yet another embodiment, the native sequence PRO245 polypeptideis a mature or full-length native sequence PRO245 polypeptide comprisingamino acids 1 to 312 of FIG. 11 (SEQ ID NO: 9).

[0083] The “PRO301 or PRO362 extracellular domain” or “PRO301 or PRO362ECD” refers to a form of the PRO301 or PRO362 polypeptide, respectively,which is essentially free of the transmembrane and cytoplasmic domainsof the respective full length molecules. Ordinarily, PRO301 ECD orPRO362 ECD will have less than 1% of such transmembrane and/orcytoplasmic domains and preferably, will have less than 0.5% of suchdomains. Optionally, PRO301 polypeptide ECD will comprise amino acidresidues about 28 to about 235 of FIG. 2 (SEQ ID NO: 1), while PRO362polypeptide ECD will comprise amino acid residues 1 to X of FIG. 3 (SEQID NO: 2), where X is any amino acid from 271-280. It will be understoodthat any transmembrane domain identified for the PRO301 or PRO362polypeptides of the present invention is identified pursuant to criteriaroutinely employed in the art for identifying that type of hydrophobicdomain. The exact boundaries of a transmembrane domain may vary but mostlikely by no more than about 5 amino acids at either end of the domainas initially identified. Accordingly, the PRO301 or PRO362 polypeptideECD may optionally comprise amino acids 1 to X of FIG. 3 (SEQ ID NO: 2),wherein X is any one of amino acid residues 271 to 280 of FIG. 3 (SEQ IDNO: 2).

[0084] “PRO301 variant” or “PRO245 variant” means an active PRO301 asdefined below having at least about 80% amino acid sequence identity to(a) a DNA molecule encoding a PRO301 polypeptide, with or without itsnative signal sequence, with or without the initiating methionine, withor without the potential transmembrane domain, and with or without theintracellular domain or (b) the complement of the DNA molecule of (a).In a particular embodiment, the PRO301 variant has at least about 80%amino acid sequence homology with the PRO301 having the deduced aminoacid sequence shown in FIG. 1 (SEQ ID NO: 1) for a full-length nativesequence PRO301. Such PRO301 variants include, for instance, PRO301polypeptides wherein one or more amino acid residues are added, ordeleted, at the N- or C-terminus of the sequence of FIG. 2 (SEQ ID NO:1). Preferably, the nucleic acid or amino acid sequence identity is atleast about 85%, more preferably at least about 90%, and even morepreferably at least about 95%.

[0085] “PRO362 variant” means an active PRO362 polypeptide as definedbelow having at least about 80% amino acid sequence identity with thePRO362 polypeptide having the deduced amino acid sequence shown in FIG.3 (SEQ ID NO: 2) for a full-length native sequence PRO362 polypeptide.Such PRO362 polypeptide variants include, for instance, PRO362polypeptides wherein one or more amino acid residues are added, ordeleted, at the N- or C- terminus of the sequence of FIG. 3 (SEQ ID NO:2). Ordinarily, a PRO362 polypeptide variant will have at least about80% amino acid sequence identity, preferably at least about 85% aminoacid sequence identity, more preferably at least about 90% amino acidsequence identity and even more preferably at least about 95% amino acidsequence identity with the amino acid sequence of FIG. 3 (SEQ ID NO: 2).

[0086] “Percent (%) amino acid sequence identity” with respect to thePRO301, PRO362 or PRO245 sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the PRO301, PRO362 or PRO245sequence, respectively, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared.

[0087] “Percent (%) nucleic acid sequence identity” with respect to thePRO301-, PRO362- or PRO245-encoding sequences identified herein (e.g.,DNA40628, DNA45416, DNA35638) is defined as the percentage ofnucleotides in a candidate sequence that are identical with thenucleotides in the PRO301-, PRO362- or PRO245-encoding sequence,respectively, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent nucleic acid sequence identity canbe achieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

[0088] “Isolated,” when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO301 naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

[0089] An “isolated” DNA40628 nucleic acid molecule is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the DNA40628 nucleic acid. An isolated DNA40628nucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated DNA40628 nucleic acid molecules thereforeare distinguished from the DNA40628 nucleic acid molecule as it existsin natural cells. However, an isolated DNA40628 nucleic acid moleculeincludes DNA40628 nucleic acid molecules contained in cells thatordinarily express DNA40628 where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

[0090] An “isolated” PRO301-PRO362- or PRO245- polypeptide encodingnucleic acid molecule is a nucleic acid molecule that is identified andseparated from at least one contaminant nucleic acid molecule with whichit is ordinarily associated in the natural source of the PRO301-PRO362-or PRO245 polypeptide encoding nucleic acid. An isolated PRO301-PRO362-or PRO245 polypeptide encoding nucleic acid molecule is other than inthe form or setting in which it is found in nature. IsolatedPRO301-PRO362- or PRO245 polypeptide encoding nucleic acid moleculestherefore are distinguished from the DNA40628 nucleic acid molecule asit exists in natural cells. However, an isolated PRO301-PRO362- orPRO245 polypeptide encoding nucleic acid molecule includesPRO301-PRO362- or PRO245 polypeptide encoding nucleic acid moleculescontained in cells that ordinarily express PRO301-PRO362- or PRO245polypeptide encoding where, for example, the nucleic acid molecule is ina chromosomal location different from that of natural cells.

[0091] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0092] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0093] The term “antibody” is used in the broadest sense andspecifically covers single anti-PRO301, anti-PRO362 or anti-PRO245monoclonal antibodies (including agonist, antagonist, and neutralizingantibodies) and anti-PRO301, anti-PRO362 or anti-PRO245 antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

[0094] “Active” or “activity” for the purposes herein refers to form(s)of PRO301, PRO362 or PRO245 which retain the biologic and/or immunologicactivities of native or naturally-occurring PRO301. A preferred activityis the ability to bind to and affect, e.g., block or otherwise modulate,an activity of antigen binding. The activity preferably involves theregulation, activity of cancer and or viral associated antigens.

[0095] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

[0096] “Stringent conditions” or “high stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42C; or (3) employ50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC sodium chloride/sodiumcitrate) and 50% formamide at 55C, followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

[0097] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

[0098] The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a polypeptide of the invention fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the polypeptide to which itis fused. The tag polypeptide preferably also is fairly unique so thatthe antibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

[0099] “Active” or “activity” in the context of variants of thepolypeptide of the invention refers to form(s) of proteins of theinvention which retain the biologic and/or immunologic activities of anative or naturally-occurring polypeptide of the invention.

[0100] “Biological activity” in the context of an antibody or anothermolecule that can be identified by the screening assays disclosed herein(e.g. an organic or inorganic small molecule, peptide, etc.) is used torefer to the ability of such molecules to induce or inhibit infiltrationof inflammatory cells into a tissue, to stimulate or inhibit T-cellproliferation and to stimulate or inhibit lymphokine release by cells.Another preferred activity is increased vascular permeability or theinhibition thereof.

[0101] The term “antagonist” is used in the broadest sense, and includesany molecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native polypeptide of the invention disclosedherein. In a similar manner, the term “agonist” is used in the broadestsense and includes any molecule that mimics a biological activity of anative polypeptide of the invention disclosed herein. Suitable agonistor antagonist molecules specifically include agonist or antagonistantibodies or antibody fragments, fragments or amino acid sequencevariants of native polypeptides of the invention, peptides, smallorganic molecules, etc.

[0102] A “small molecule” is defined herein to have a molecular weightbelow about 600 daltons.

[0103] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.The term “antibody” is used in the broadest sense and specificallycovers, without limitation, intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g. bispecific antibodies) formedfrom at least two intact antibodies, and antibody fragments so long asthey exhibit the desired biological activity.

[0104] “Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains.

[0105] The term “variable” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three segments called complementarity-determiningregions (CDRs) or hypervariable regions both in the light-chain and theheavy-chain variable domains. The more highly conserved portions ofvariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a beta-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the beta-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen-binding site of antibodies (see Kabat etal., NIH Publ. No.91-3242, Vol. I, pages 647-669 (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

[0106] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10):1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0107] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment. The designation “Fc”reflects the ability to crystallize readily. Pepsin treatment yields anF(ab′)₂ fragment that has two antigen-combining sites and is stillcapable of cross-linking antigen.

[0108] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0109] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

[0110] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa (κ) and lambda (λ), based on the amino acid sequences oftheir constant domains.

[0111] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

[0112] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, ie., the individual antibodies comprising the population areidentical except for possible naturally occurring mutations that may bepresent in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., Nature, 256:495 [1975], or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 [1991] and Marks et al., J. Mol. Boil., 222: 581-597 (1991),for example. See also U.S. Pat. Nos. 5,750,373, 5,571,698, 5,403,484 and5,223,409 which describe the preparation of antibodies using phagemidand phage vectors.

[0113] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).

[0114] “Humanized” forms of non-human (e.g., murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which several or all residuesfrom a complementarity-determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinity,and capacity. In some instances, certain Fv framework region (FR)residues of the human immunoglobulin can also be replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and maximize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525 (1986); Reichmann et al, Nature, 332:323-329 [1988]; and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).The humanized antibody includes a “primatized” antibody where theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.Antibodies containing residues from Old World monkeys are also possiblewithin the invention. See, for example, U.S. Pat. Nos. 5,658,570;5,693,780; 5,681,722; 5,750,105; and 5,756,096.

[0115] “Single-chain Fv” or “sFv” antibody fragments comprise the VH andVL domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

[0116] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0117] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the compound of theinvention will be purified (1) to greater than 95% by weight of thecompound as determined by the Lowry method, and most preferably morethan 99% by weight, (2) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or, preferably,silver stain. Isolated compound, e.g. antibody or polypeptide, includesthe compound in situ within recombinant cells since at least onecomponent of the compound's natural environment will not be present.Ordinarily, however, isolated compound will be prepared by at least onepurification step.

[0118] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to thecompound, e.g. antibody or polypeptide, so as to generate a “labeled”compound. The label may be detectable by itself (e.g. radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

[0119] By “solid phase” is meant a non-aqueous matrix to which thecompound of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

[0120] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant which is useful for delivery ofa drug (such as the anti-ErbB2 antibodies disclosed herein and,optionally, a chemotherapeutic agent) to a mammal. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes.

[0121] As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

II. Compositions and Methods of the Invention

[0122] A. Preparation of the PRO301, PRO362 or PRO245 Polypeptides

[0123] 1. Full-length PRO301, PRO362 or PRO245 Polypeptides

[0124] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO301, PRO362 or PRO245. In particular, Applicants haveidentified and isolated cDNA encoding a PRO301, PRO362 or PRO245polypeptide, as disclosed in further detail in the Examples below. UsingBLAST and FatA sequence alignment computer programs, Applicants foundthat a full-length native sequence PRO301 (FIG. 2, SEQ ID NO: 1), PRO362(FIG. 3, SEQ ID NO: 3) and PRO245 (FIG. 11, SEQ ID NO: 9) havesignificant homology to both A33 antigen and JAM. (See FIGS. 1, 12-18).Accordingly, it is presently believed that PRO301 disclosed in thepresent application is a newly identified member of the A33 antigenprotein family and may be associated with inflammatory disorders such asinflammatory bowel disease as well as human neoplastic diseases such ascolorectal cancer.

[0125] 2. PRO301, PRO362 or PRO245 Variants

[0126] In addition to the full-length native sequence PRO301, PRO362 orPRO245 described herein, it is contemplated that PRO301, PRO362 orPRO245 variants can be prepared. PRO301, PRO362 or PRO245 variants canbe prepared by introducing appropriate nucleotide changes into thePRO301, PRO362 or PRO245 DNA, respectively, or by synthesis of thedesired PRO301, PRO362 or PRO245 polypeptides. Those skilled in the artwill appreciate that amino acid changes may alter post-translationalprocesses of the PRO301, PRO362 or PRO245, such as changing the numberor position of glycosylation sites or altering the membrane anchoringcharacteristics.

[0127] Variations in the native full-length sequence PRO301, PRO362 orPRO245 or in various domains of the PRO301, PRO362 or PRO245 describedherein, can be made, for example, using any of the techniques andguidelines for conservative and non-conservative mutations set forth,for instance, in U.S. Pat. No. 5,364,934. Variations may be asubstitution, deletion or insertion of one or more codons encoding thePRO301, PRO362 or PRO245 that results in a change in the amino acidsequence of the PRO301, PRO362 or PRO245 as compared with the nativesequence PRO301, PRO362 or PRO245. Optionally the variation is bysubstitution of at least one amino acid with any other amino acid in oneor more of the domains of the PRO301, PRO362 or PRO245. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO301, PRO362 or PRO245 with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of 1 to 5 amino acids. The variation allowed may be determined bysystematically making insertions, deletions or substitutions of aminoacids in the sequence and testing the resulting variants for activity inthe in vitro assay described in the Examples below.

[0128] The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO301 variant DNA.

[0129] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions [Creighton,The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used.

[0130] 3. Modifications of PRO301, PRO362 or PRO245

[0131] Covalent modifications of PRO301, PRO362 or PRO245 are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of the PRO301, PRO362 orPRO245 with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues of thePRO301, PRO362 or PRO245. Derivatization with bifunctional agents isuseful, for instance, for crosslinking PRO301 to a water-insolublesupport matrix or surface for use in the method for purifyinganti-PRO301 antibodies, and vice-versa. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis-(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0132] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains [T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0133] Another type of covalent modification of the PRO301, PRO362 orPRO245 polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequencePRO301, PRO362 or PRO245, and/or adding one or more glycosylation sitesthat are not present in the native sequence PRO301, PRO362 or PRO245,and/or alteration of the ratio and/or composition of the sugar residuesattached to the glycosylation site(s).

[0134] Addition of glycosylation sites to the PRO301, PRO362 orPRO245polypeptide may be accomplished by altering the amino acidsequence. The alteration may be made, for example, by the addition of,or substitution by, one or more serine or threonine residues to thenative sequence PRO301, PRO362 or PRO245 (for O-linked glycosylationsites). The PRO301, PRO362 or PRO245 amino acid sequence may optionallybe altered through changes at the DNA level, particularly by mutatingthe DNA encoding the PRO301, PRO362 or PRO245 polypeptide at preselectedbases such that codons are generated that will translate into thedesired amino acids.

[0135] Another means of increasing the number of carbohydrate moietieson the PRO301, PRO362 or PRO245 polypeptide is by chemical or enzymaticcoupling of glycosides to the polypeptide. Such methods are described inthe art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0136] Removal of carbohydrate moieties present on the PRO301, PRO362 orPRO245 polypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

[0137] Another type of covalent modification of PRO301, PRO362 or PRO245comprises linking the PRO301, PRO362 or PRO245 polypeptide to one of avariety of nonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

[0138] The PRO301, PRO362 or PRO245 of the present invention may also bemodified in a way to form a chimeric molecule comprising PRO301, PRO362or PRO245 fused to another, heterologous polypeptide or amino acidsequence. In one embodiment, such a chimeric molecule comprises a fusionof the PRO301, PRO362 or PRO245 with a tag polypeptide which provides anepitope to which an anti-tag antibody can selectively bind. The epitopetag is generally placed at the amino- or carboxyl-terminus of thePRO301, PRO362 or PRO245. The presence of such epitope-tagged forms ofthe PRO301, PRO362 or PRO245 can be detected using an antibody againstthe tag polypeptide. Also, provision of the epitope tag enables thePRO301, PRO362 or PRO245 to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag. In an alternative embodiment, the chimeric moleculemay comprise a fusion of the PRO301, PRO362 or PRO245 with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule, such a fusion could be to the Fcregion of an IgG molecule.

[0139] Various tag polypeptides and their respective antibodies are wellknown in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3 C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

[0140] 4. Production and Isolation of PRO301, PRO362 or PRO245

[0141] The description below relates primarily to production of PRO301,PRO362 or PRO245 by culturing cells transformed or transfected with avector containing PRO301, PRO362 or PRO245 nucleic acid. It is, ofcourse, contemplated that alternative methods, which are well known inthe art, may be employed to prepare PRO301, PRO362 or PRO245. Forinstance, the PRO301, PRO362 or PRO245 sequence, or portions thereof,may be produced by direct peptide synthesis using solid-phase techniques[see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W. H. FreemanCo., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the PRO301, PRO362 or PRO245 may be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce the full-length PRO301, PRO362 or PRO245.

[0142] a. Isolation of DNA Encoding PRO301, PRO362 or PRO245

[0143] DNA encoding PRO301, PRO362 or PRO245 may be obtained from a cDNAlibrary prepared from tissue believed to possess the PRO301, PRO362 orPRO245 mRNA and to express it at a detectable level. Accordingly, humanPRO301, PRO362 or PRO245 DNA can be conveniently obtained from a cDNAlibrary prepared from human tissue, such as described in the Examples.The PRO301-, PRO362- or PRO245-encoding gene may also be obtained from agenomic library or by oligonucleotide synthesis.

[0144] Libraries can be screened with probes (such as antibodies to thePRO301, PRO362 or PRO245 or oligonucleotides of at least about 20-80bases) designed to identify the gene of interest or the protein encodedby it. Screening the cDNA or genomic library with the selected probe maybe conducted using standard procedures, such as described in Sambrook etal., Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding PRO301, PRO362 or PRO245 is to use PCR methodology [Sambrook etal., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (ColdSpring Harbor Laboratory Press, 1995)].

[0145] The Examples below describe techniques for screening a cDNAlibrary. The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The oligonucleotide is preferably labeled such that it can bedetected upon hybridization to DNA in the library being screened.Methods of labeling are well known in the art, and include the use ofradiolabels like ³²p-labeled ATP, biotinylation or enzyme labeling.Hybridization conditions, including moderate stringency and highstringency, are provided in Sambrook et al., supra.

[0146] Sequences identified in such library screening methods can becompared and aligned to other known sequences deposited and available inpublic databases such as GenBank or other private sequence databases.Sequence identity (at either the amino acid or nucleotide level) withindefined regions of the molecule or across the full-length sequence canbe determined through sequence alignment using computer softwareprograms such as BLAST, BLAST-2, ALIGN, DNAstar, and INHERIT whichemploy various algorithms to measure homology.

[0147] Nucleic acid having protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of MRNAthat may not have been reverse-transcribed into cDNA.

[0148] b. Selection and Transformation of Host Cells

[0149] Host cells are transfected or transformed with expression orcloning vectors described herein for PRO301, PRO362 or PRO245 productionand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general., principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: A Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0150] Methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ and electroporation. Depending on the hostcell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes or other cells that contain substantialcell-wall barriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

[0151] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).

[0152] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forPRO301-, PRO362- or PRO245-encoding vectors. Saccharomyces cerevisiae isa commonly used lower eukaryotic host microorganism.

[0153] Suitable host cells for the expression of glycosylated PRO301,PRO362 or PRO245 are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells. Examples of useful mammalianhost cell lines include Chinese hamster ovary (CHO) and COS cells. Morespecific examples include monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlauband Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertolicells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mousemammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriatehost cell is deemed to be within the skill in the art.

[0154] C. Selection and Use of a Replicable Vector

[0155] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO301,PRO362 or PRO245 may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

[0156] The PRO301, PRO362 or PRO245 may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the PRO30 1, PRO362 or PRO245 DNA that isinserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces”—factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

[0157] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 21μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells.

[0158] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0159] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thePRO301. PRO362 or PRO245 nucleic acid, such as DHFR or thymidine kinase.An appropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

[0160] Expression and cloning vectors usually contain a promoteroperably linked to the PRO301, PRO362 or PRO245 nucleic acid sequence todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known. Promoters suitable for use with prokaryotichosts include the β-lactamase and lactose promoter systems [Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingPRO301, PRO362 or PRO245.

[0161] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0162] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0163] PRO301, PRO362 or PRO245 transcription from vectors in mammalianhost cells is controlled, for example, by promoters obtained from thegenomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, and from heat-shock promoters, provided such promoters arecompatible with the host cell systems.

[0164] Transcription of a DNA encoding the PRO301, PRO362 or PRO245 byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Many enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein, and insulin). Typically, however, onewill use an enhancer from a eukaryotic cell virus. Examples include theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO301, PRO362 or PRO245 coding sequence, but is preferably located at asite 5′ from the promoter.

[0165] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal., human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding PRO301, PRO362 or PRO245.

[0166] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of PRO301, PRO362 or PRO245 in recombinantvertebrate cell culture are described in Gething et al., Nature,293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060;and EP 117,058.

[0167] d. Detecting Gene Amplification/Expression

[0168] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0169] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO301, PRO362 or PRO245 polypeptide or against a synthetic peptidebased on the DNA sequences provided herein or against exogenous sequencefused to PRO301, PRO362 or PRO245 DNA and encoding a specific antibodyepitope.

[0170] e. Purification of Polypeptide

[0171] Forms of PRO301, PRO362 or PRO245 may be recovered from culturemedium or from host cell lysates. If membrane-bound, it can be releasedfrom the membrane using a suitable detergent solution (e.g. Triton-X100) or by enzymatic cleavage. Cells employed in expression of PRO301,PRO362 or PRO245 can be disrupted by various physical or chemical means,such as freeze-thaw cycling, sonication, mechanical disruption, or celllysing agents.

[0172] It may be desired to purify PRO301, PRO362 or PRO245 fromrecombinant cell proteins or polypeptides. The following procedures areexemplary of suitable purification procedures: by fractionation on anion-exchange column; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; protein A Sepharosecolumns to remove contaminants such as IgG; and metal chelating columnsto bind epitope-tagged forms of the PRO301, PRO362 or PRO245. Variousmethods of protein purification may be employed and such methods areknown in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular PRO301, PRO362 or PRO245 produced.

[0173] 2. Tissue Distribution

[0174] The location of tissues expressing the polypeptides of theinvention can be identified by determining mRNA expression in varioushuman tissues. The location of such genes provides information aboutwhich tissues are most likely to be affected by the stimulating andinhibiting activities of the polypeptides of the invention. The locationof a gene in a specific tissue also provides sample tissue for theactivity blocking assays discussed below.

[0175] Gene expression in various tissues may be measured byconventional Southern blotting, Northern blotting to quantitate thetranscription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205[1980]), dot blotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequences provided herein.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes.

[0176] Gene expression in various tissues, alternatively, may bemeasured by immunological methods, such as immunohistochemical stainingof tissue sections and assay of cell culture or body fluids, toquantitate directly the expression of gene product. Antibodies usefulfor immunohistochemical staining and/or assay of sample fluids may beeither monoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceof a polypeptide of the invention or against a synthetic peptide basedon the DNA sequences encoding the polypeptide of the invention oragainst an exogenous sequence fused to a DNA encoding a polypeptide ofthe invention and encoding a specific antibody epitope. Generaltechniques for generating antibodies, and special protocols for Northernblotting and in situ hybridization are provided below.

[0177] 3. Antibody Binding Studies

[0178] The activity of the polypeptides of the invention can be furtherverified by antibody binding studies, in which the ability ofanti-PRO301, anti-PRO362 or anti-PRO245 antibodies to inhibit the effectof the PRO301, PRO362 or PRO245 polypeptides on tissue cells is tested.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies, the preparation of whichwill be described hereinbelow.

[0179] Antibody binding studies may be carried out in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

[0180] Competitive binding assays rely on the ability of a labeledstandard to compete with the test sample analyte for binding with alimited amount of antibody. The amount of target protein in the testsample is inversely proportional to the amount of standard that becomesbound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies preferably are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

[0181] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three-part complex. See, e.g., U.S. Pat No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

[0182] For immunohistochemistry, the tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin, for example.

[0183] 4. Cell-Based Assays

[0184] Cell-based assays and animal models for immune related diseasescan be used to further understand the relationship between the genes andpolypeptides identified herein and the development and pathogenesis ofimmune related disease.

[0185] In a different approach, cells of a cell type known to beinvolved in a particular immune related disease are transfected with thecDNAs described herein, and the ability of these cDNAs to stimulate orinhibit immune function is analyzed. Suitable cells can be transfectedwith the desired gene, and monitored for immune function activity. Suchtransfected cell lines can then be used to test the ability of poly- ormonoclonal antibodies or antibody compositions to inhibit or stimulateimmune function, for example to modulate T-cell proliferation orinflammatory cell infiltration. Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of immune related diseases.

[0186] In addition, primary cultures derived from transgenic animals (asdescribed below) can be used in the cell-based assays herein, althoughstable cell lines are preferred. Techniques to derive continuous celllines from transgenic animals are well known in the art (see, e.g. Smallet al., Mol. Cell. Biol. 5,642-648 [1985]).

[0187] One suitable cell based assay is the mixed lymphocyte reaction(MLR). Current Protocols in Immunology, unit 3.12; edited by J EColigan, A M Kruisbeek, D H Marglies, E M Shevach, W Strober, NationalInstitutes of Health, Published by John Wiley & Sons, Inc. In thisassay, the ability of a test compound to stimulate the proliferation ofactivated T cells is assayed. A suspension of responder T cells iscultured with allogeneic stimulator cells and the proliferation of Tcells is measured by uptake of tritiated thymidine. This assay is ageneral measure of T cell reactivity. Since the majority of T cellsrespond to and produce IL-2 upon activation, differences inresponsiveness in this assay in part reflect differences in IL-2production by the responding cells. The MLR results can be verified by astandard lymphokine (IL-2) detection assay. Current Protocols inImmunology, supra, 3.15, 6.3.

[0188] A proliferative T cell response in an MLR assay may be due to amitogenic response or may be due to a stimulatory response by the Tcells. Additional verification of the T cell stimulatory activity of thepolypeptides of the invention can be obtained by a costimulation assay.T cell activation requires an antigen specific signal mediated throughthe major histocompatability complex (MHC) and a costimulatory signalmediated through a second ligand binding interaction, for example, theB7(CD80, CD86)/CD28 binding interaction. CD28 crosslinking increaseslymphokine secretion by activated T cells. T cell activation has bothnegative and positive controls through the binding of ligands which havea negative or positive effect. CD28 and CTLA-4 are related glycoproteinsin the Ig superfamily which bind to B7. CD28 binding to B7 has apositive costimulation effect of T cell activation; conversely, CTLA-4binding to B7 has a negative T cell deactivating effect. Chambers, C. A.and Allison, J. P., Curr. Opin. Immunol. (1997) 9:396. Schwartz, R. H.,Cell (1992) 71:1065; Linsey, P. S. and Ledbetter, J. A., Annu. Rev.Immunol. (1993) 11:191; June, C. H. et al., Immunol. Today (1994)15:321; Jenkins, M. K., Immunity (1994) 1:405. In a costimulation assay,the polypeptides of the invention are assayed for T cell costimulatoryor inhibitory activity.

[0189] Polypeptides of the invention, as well as other compounds of theinvention, which are stimulators (costimulators) of T cellproliferation, as determined by MLR and costimulation assays,forexample, are useful in treating immune related diseases characterized bypoor, suboptimal or inadequate immune function. These diseases aretreated by stimulating the proliferation and activation of T cells (andT cell mediated immunity) and enhancing the immune response in a mammalthrough administration of a stimulatory compound, such as thestimulating polypeptides of the invention. The stimulating polypeptidemay be a PRO301, PRO362 or PRO245 polypeptide or an agonist antibodytherefor. Immunoadjuvant therapy for treatment of tumors, described inmore detail below, is an example of this use of the stimulatingcompounds of the invention. Antibodies which bind to inhibitorypolypeptides function to enhance the immune response by removing theinhibitory effect of the inhibiting polypeptides. This effect is seen inexperiments using anti-CTLA-4 antibodies which enhance T cellproliferation, presumably by removal of the inhibitory signal caused byCTLA-4 binding. Walunas, T. L. et al., Immunity (1994) 1:405. This useis also validated in experiments with 4-1BB glycoprotein, a member ofthe tumor necrosis factor receptor family which binds to a ligand(4-1BBL) expressed on primed T cells and signals T cell activation andgrowth. Alderson, M. E. et al., J. Immunol. (1994) 24:2219. Inhibitionof 4-1BB binding by treatment with an anti-4-1BB antibody increases theseverity of graft-versus-host disease and may be used to eradicatetumors. Hellstrom, I. and Hellstrom, K. E., Crit. Rev. Immunol. (1998)18:1.

[0190] On the other hand, polypeptides of the invention, as well asother compounds of the invention, which are inhibitors of T cellproliferation/activation and/or lymphokine secretion, can be directlyused to suppress the immune response. These compounds are useful toreduce the degree of the immune response and to treat immune relateddiseases characterized by a hyperactive, superoptimal., or autoimmuneresponse. Alternatively, antibodies which bind to the stimulatingpolypeptides of the invention and block the stimulating effect of thesemolecules can be used to suppress the T cell mediated immune response byinhibiting T cell proliferation/activation and/or lymphokine secretion.Blocking the stimulating effect of the polypeptides suppresses theimmune response of the mammal.

[0191] 5. Animal Models

[0192] The results of the cell based in vitro assays can be furtherverified using in vivo animal models and assays for T-cell function. Avariety of well known animal models can be used to further understandthe role of the genes identified herein in the development andpathogenesis of immune related disease, and to test the efficacy ofcandidate therapeutic agents, including antibodies, and otherantagonists of the native polypeptides, including small moleculeantagonists. The in vivo nature of such models makes them particularlypredictive of responses in human patients. Animal models of immunerelated diseases include both non-recombinant and recombinant(transgenic) animals. Non-recombinant animal models include, forexample, rodent, e.g., murine models. Such models can be generated byintroducing cells into syngeneic mice using standard techniques, e.g.subcutaneous injection, tail vein injection, spleen implantation,intraperitoneal implantation, implantation under the renal capsule, etc.

[0193] Contact hypersensitivity is a simple in vivo assay of cellmediated immune function. In this procedure, epidermal cells are exposedto exogenous haptens which give rise to a delayed type hypersensitivityreaction which is measured and quantitated. Contact sensitivity involvesan initial sensitizing phase followed by an elicitation phase. Theelicitation phase occurs when the epidermal cells encounter an antigento which they have had previous contact. Swelling and inflammationoccur, making this an excellent model of human allergic contactdermatitis. A suitable procedure is described in detail in CurrentProtocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc., 1994,unit 4.2. See also Grabbe, S. and Schwarz, T, Immun. Today 19(1):37-44(1998).

[0194] Graft-versus-host disease occurs when immunocompetent cells aretransplanted into immunosuppressed or tolerant patients. The donor cellsrecognize and respond to host antigens. The response can vary from lifethreatening severe inflammation to mild cases of diarrhea and weightloss. Graft-versus-host disease models provide a means of assessing Tcell reactivity against MHC antigens and minor transplant antigens. Asuitable procedure is described in detail in Current Protocols inImmunology, supra, unit 4.3.

[0195] An animal model for skin allograft rejection is a means oftesting the ability of T cells to mediate in vivo tissue destructionwhich is indicative of and a measure of their role in anti-viral andtumor immunity. The most common and accepted models use murine tail-skingrafts. Repeated experiments have shown that skin allograft rejection ismediated by T cells, helper T cells and killer-effector T cells, and notantibodies. Auchincloss, H. Jr. and Sachs, D. H., FundamentalImmunology, 2nd ed., W. E. Paul ed., Raven Press, NY, 1989, 889-992. Asuitable procedure is described in detail in Current Protocols inImmunology, supra, unit 4.4. Other transplant rejection models which canbe used to test the compounds of the invention are the allogeneic hearttransplant models described by Tanabe, M. et al., Transplantation (1994)58:23 and Tinubu, S. A. et al., J. Immunol. (1994) 4330-4338.

[0196] Animal models for delayed type hypersensitivity provides an assayof cell mediated immune function as well. Delayed type hypersensitivityreactions are a T cell mediated in vivo immune response characterized byinflammation which does not reach a peak until after a period of timehas elapsed after challenge with an antigen. These reactions also occurin tissue specific autoimmune diseases such as multiple sclerosis (MS)and experimental autoimmune encephalomyelitis (EAE, a model for MS). Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.5.

[0197] EAE is a T cell mediated autoimmune disease characterized by Tcell and mononuclear cell inflammation and subsequent demyelination ofaxons in the central nervous system. EAE is generally considered to be arelevant animal model for MS in humans. Bolton, C., Multiple Sclerosis(1995) 1:143. Both acute and relapsing-remitting models have beendeveloped. The compounds of the invention can be tested for T cellstimulatory or inhibitory activity against immune mediated demyelinatingdisease using the protocol described in Current Protocols in Immunology,above, units 15.1 and 15.2. See also the models for myelin disease inwhich oligodendrocytes or Schwann cells are grafted into the centralnervous system as described in Duncan, I. D. et al., Molec. Med. Today(1997) 554-561.

[0198] An animal model for arthritis is collagen-induced arthritis. Thismodel shares clinical, histological and immunological characteristics ofhuman autoimmune rheumatoid arthritis and is an acceptable model forhuman autoimmune arthritis. Mouse and rat models are characterized bysynovitis, erosion of cartilage and subchondral bone. The compounds ofthe invention can be tested for activity against autoimmune arthritisusing the protocols described in Current Protocols in Immunology, above,units 15.5. See also the model using a monoclonal antibody to CD18 andVLA-4 integrins described in Issekutz, A. C. et al., Immunology (1996)88:569.

[0199] A model of asthma has been described in which antigen-inducedairway hyper-reactivity, pulmonary eosinophilia and inflammation areinduced by sensitizing an animal with ovalbumin and then challenging theanimal with the same protein delivered by aerosol. Several animal models(guinea pig, rat, non-human primate) show symptoms similar to atopicasthma in humans upon challenge with aerosol antigens. Murine modelshave many of the features of human asthma. Suitable procedures to testthe compounds of the invention for activity and effectiveness in thetreatment of asthma are described by Wolyniec, W. W. et al., Am. J.Respir. Cell Mol. Biol. (1998) 18:777 and the references cited therein.

[0200] Additionally, the compounds of the invention can be tested onanimal models for psoriasis like diseases. Evidence suggests a T cellpathogenesis for psoriasis. The compounds of the invention can be testedin the scid/scid mouse model described by Schon, M. P. et al, Nat. Med.(1997) 3:183, in which the mice demonstrate histopathologic skin lesionsresembling psoriasis. Another suitable model is the human skin/scidmouse chimera prepared as described by Nickoloff, B. J. et al., Am. J.Path. (1995) 146:580.

[0201] Recombinant (transgenic) animal models can be engineered byintroducing the coding portion of the genes identified herein into thegenome of animals of interest, using standard techniques for producingtransgenic animals. Animals that can serve as a target for transgenicmanipulation include, without limitation, mice, rats, rabbits, guineapigs, sheep, goats, pigs, and non-human primates, e.g. baboons,chimpanzees and monkeys. Techniques known in the art to introduce atransgene into such animals include pronucleic microinjection (Hoppe andWanger, U.S. Pat. No. 4,873,191); retrovirus-mediated gene transfer intogerm lines (e.g., Van der Putten et al., Proc. Natl. Acad. Sci USA 82,6148-615 [1985]); gene targeting in embryonic stem cells (Thompson etal., Cell 56, 313-321 [1989]); electroporation of embryos (Lo, Mol. Cel.Biol. 3, 1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano etal., Cell 57, 717-73 [1989]). For review, see, for example, U.S. Pat.No. 4,736,866.

[0202] For the purpose of the present invention, transgenic animalsinclude those that carry the transgene only in part of their cells(“mosaic animals”). The transgene can be integrated either as a singletransgene, or in concatamers, e.g., head-to-head or head-to-tailtandems. Selective introduction of a transgene into a particular celltype is also possible by following, for example, the technique of Laskoet al., Proc. Natl. Acad. Sci. USA 89, 623-636 (1992).

[0203] The expression of the transgene in transgenic animals can bemonitored by standard techniques. For example, Southern blot analysis orPCR amplification can be used to verify the integration of thetransgene. The level of mRNA expression can then be analyzed usingtechniques such as in situ hybridization, Northern blot analysis, PCR,or immunocytochemistry.

[0204] The animals may be further examined for signs of immune diseasepathology, for example by histological examination to determineinfiltration of immune cells into specific tissues. Blocking experimentscan also be performed in which the transgenic animals are treated withthe compounds of the invention to determine the extent of the T cellproliferation stimulation or inhibition of the compounds. In theseexperiments, blocking antibodies which bind to the polypeptide of theinvention, prepared as described above, are administered to the animaland the effect on immune function is determined.

[0205] Alternatively, “knock out” animals can be constructed which havea defective or altered gene encoding a polypeptide identified herein, asa result of homologous recombination between the endogenous geneencoding the polypeptide and altered genomic DNA encoding the samepolypeptide introduced into an embryonic cell of the animal. Forexample, cDNA encoding a particular polypeptide can be used to clonegenomic DNA encoding that polypeptide in accordance with establishedtechniques. A portion of the genomic DNA encoding a particularpolypeptide can be deleted or replaced with another gene, such as a geneencoding a selectable marker which can be used to monitor integration.Typically, several kilobases of unaltered flanking DNA (both at the 5′and 3′ ends) are included in the vector [see e.g., Thomas and Capecchi,Cell, 51:503 (1987) for a description of homologous recombinationvectors]. The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected [see e.g.,Li et al., Cell, 69:915 (1992)]. The selected cells are then injectedinto a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the polypeptide.

[0206] 6. Immuno Adjuvant Therapy

[0207] In one embodiment, compounds of the invention having animmunostimulatory effect can be used in immunoadjuvant therapy for thetreatment of tumors (cancer). It is now well established that T cellsrecognize human tumor specific antigens. One group of tumor antigens,encoded by the MAGE, BAGE and GAGE families of genes, are silent in alladult normal tissues, but are expressed in significant amounts intumors, such as melanomas, lung tumors, head and neck tumors, andbladder carcinomas. DeSmet, C. et al., (1996) Proc. Natl. Acad. Sci.USA, 93:7149. It has been shown that costimulation of T cells inducestumor regression and an antitumor response both in vitro and in vivo.Melero, I. et al., Nature Medicine (1997) 3:682; Kwon, E. D. et al.,Proc. Natl. Acad. Sci. USA (1997) 94:8099; Lynch, D. H. et al., NatureMedicine (1997) 3:625; Finn, O. J. and Lotze, M. T., J. Immunol. (1998)21:114. The stimulatory compounds of the invention can be administeredas adjuvants, alone or together with a growth regulating agent,cytotoxic agent or chemotherapeutic agent, to stimulate T cellproliferation/activation and an antitumor response to tumor antigens.The growth regulating, cytotoxic, or chemotherapeutic agent may beadministered in conventional amounts using known administration regimes.Immunostimulating activity by the compounds of the invention allowsreduced amounts of the growth regulating, cytotoxic, or chemotherapeuticagents thereby potentially lowering the toxicity to the patient.

[0208] Cancer is characterized by the increase in the number ofabnormal., or neoplastic, cells derived from a normal tissue whichproliferate to form a tumor mass, the invasion of adjacent tissues bythese neoplastic tumor cells, and the generation of malignant cellswhich eventually spread via the blood or lymphatic system to regionallymph nodes and to distant sites (metastasis). In a cancerous state acell proliferates under conditions in which normal cells would not grow.Cancer manifests itself in a wide variety of forms, characterized bydifferent degrees of invasiveness and aggressiveness.

[0209] Alteration of gene expression is intimately related to theuncontrolled cell growth and de-differentiation which are a commonfeature of all cancers. The genomes of certain well studied tumors havebeen found to show decreased expression of recessive genes, usuallyreferred to as tumor suppression genes, which would normally function toprevent malignant cell growth, and/or overexpression of certain dominantgenes, such as oncogenes, that act to promote malignant growth. Each ofthese genetic changes appears to be responsible for importing some ofthe traits that, in aggregate, represent the full neoplastic phenotype(Hunter, Cell 64, 1129 [1991]; Bishop, Cell 64, 235-248 [1991]).

[0210] A well known mechanism of gene (e.g. oncogene) overexpression incancer cells is gene amplification. This is a process where in thechromosome of the ancestral cell multiple copies of a particular geneare produced. The process involves unscheduled replication of the regionof chromosome comprising the gene, followed by recombination of thereplicated segments back into the chromosome (Alitalo et al., Adv.Cancer Res. 47, 235-281 [1986]). It is believed that the overexpressionof the gene parallels gene amplification, i.e. is proportionate to thenumber of copies made.

[0211] Proto-oncogenes that encode growth factors and growth factorreceptors have been identified to play important roles in thepathogenesis of various human malignancies, including breast cancer. Forexample, it has been found that the human ErbB2 gene (erbB2, also knownas her2, or c-erbB-2), which encodes a 185-kd transmembrane glycoproteinreceptor (p185^(HER2); HER2) related to the epidermal growth factorreceptor (EGFR), is overexpressed in about 25% to 30% of human breastcancer (Slamon et al., Science 235:177-182 [1987]; Slamon et al.,Science 244:707-712 [1989]).

[0212] It has been reported that gene amplification of a protooncogen isan event typically involved in the more malignant forms of cancer, andcould act as a predictor of clinical outcome (Schwab et al., GenesChromosomes Cancer 1, 181-193 [1990]; Alitalo et al., supra). Thus,erbB2 overexpression is commonly regarded as a predictor of a poorprognosis, especially in patients with primary disease that involvesaxillary lymph nodes (Slamon et al., [1987] and [1989], supra; Ravdinand Chamness, Gene 159:19-27 [1995]; and Hynes and Stern, BiochimBiophys Acta 1198:165-184 [1994]), and has been linked to sensitivityand/or resistance to hormone therapy and chemotherapeutic regimens,including CMF (cyclophosphamide, methotrexate, and fluoruracil) andanthracyclines (Baselga et al., Oncology 11(3 Suppl 1):43-48 [1997]).However, despite the association of erbB2 overexpression with poorprognosis, the odds of HER2-positive patients responding clinically totreatment with taxanes were greater than three times those ofHER2-negative patients (Ibid). A recombinant humanized anti-ErbB2(anti-HER2) monoclonal antibody (a humanized version of the murineanti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or Herceptin7) hasbeen clinically active in patients with ErbB2-overexpressing metastaticbreast cancers that had received extensive prior anticancer therapy.(Baselga et al., J. Clin. Oncol. 14:737-744 [1996]).

[0213] 7. Screening Assays for Drug Candidates

[0214] Screening assays for drug candidates are designed to identifycompounds that bind or complex with the polypeptides encoded by thegenes identified herein or a biologically active fragment thereof, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds, including peptides, preferably soluble peptides,(poly)peptide-immunoglobulin fusions, and, in particular, antibodiesincluding, without limitation, poly- and monoclonal antibodies andantibody fragments, single-chain antibodies, anti-idiotypic antibodies,and chimeric or humanized versions of such antibodies or fragments, aswell as human antibodies and antibody fragments. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art.

[0215] All assays are common in that they call for contacting the drugcandidate with a polypeptide encoded by a nucleic acid identified hereinunder conditions and for a time sufficient to allow these two componentsto interact.

[0216] In binding assays, the interaction is binding and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, the polypeptide encoded by the gene identifiedherein or the drug candidate is immobilized on a solid phase, e.g. on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the polypeptide and drying. Alternatively, an immobilizedantibody, e.g. a monoclonal antibody, specific for the polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g. the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g. by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labeledantibody specifically binding the immobilized complex.

[0217] If the candidate compound interacts with but does not bind to aparticular protein encoded by a gene identified herein, its interactionwith that protein can be assayed by methods well known for detectingprotein-protein interactions. Such assays include traditionalapproaches, such as, cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers [Fieldsand Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA 88, 9578-9582 (1991)] as disclosed by Chevray and Nathans[Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991)]. Many transcriptionalactivators, such as yeast GAL4, consist of two physically discretemodular domains, one acting as the DNA-binding domain, while the otherone functioning as the transcription activation domain. The yeastexpression system described in the foregoing publications (generallyreferred to as the “two-hybrid system”) takes advantage of thisproperty, and employs two hybrid proteins, one in which the targetprotein is fused to the DNA-binding domain of GAL4, and another, inwhich candidate activating proteins are fused to the activation domain.The expression of a GAL1-lacZ reporter gene under control of aGAL4-activated promoter depends on reconstitution of GAL4 activity viaprotein-protein interaction. Colonies containing interactingpolypeptides are detected with a chromogenic substrate forγ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

[0218] In order to find compounds that interfere with the interaction ofa gene identified herein and other intra- or extracellular componentscan be tested, a reaction mixture is usually prepared containing theproduct of the gene and the intra- or extracellular component underconditions and for a time allowing for the interaction and binding ofthe two products. To test the ability of a test compound to inhibitbinding, the reaction is run in the absence and in the presence of thetest compound. In addition, a placebo may be added to a third reactionmixture, to serve as positive control. The binding (complex formation)between the test compound and the intra- or extracellular componentpresent in the mixture is monitored as described above. The formation ofa complex in the control reaction(s) but not in the reaction mixturecontaining the test compound indicates that the test compound interfereswith the interaction of the test compound and its reaction partner.

[0219] 8. Compositions and Methods for the Treatment of Immune RelatedDiseases

[0220] The compositions useful in the treatment of immune relateddiseases include, without limitation, antibodies, small organic andinorganic molecules, peptides, phosphopeptides, antisense and ribozymemolecules, triple helix molecules, etc. that inhibit or stimulate immunefunction, for example, T cell proliferation/activation, lymphokinerelease, or immune cell infiltration.

[0221] For example, antisense RNA and RNA molecule act to directly blockthe translation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g. between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

[0222] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. Ribozymes act by sequence-specifichybridization to the complementary target RNA, followed byendonucleolytic cleavage. Specific ribozyme cleavage sites within apotential RNA target can be identified by known techniques. For furtherdetails see, e.g. Rossi, Current Biology 4, 469-471 (1994), and PCTpublication No. WO 97/33551 (published Sep. 18, 1997).

[0223] Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g. PCTpublication No. WO 97/3355 1, supra.

[0224] These molecules can be identified by any or any combination ofthe screening assays discussed above and/or by any other screeningtechniques well known for those skilled in the art.

[0225] 9. Antibodies

[0226] Among the most promising drug candidates according to the presentinvention are antibodies and antibody fragments which may inhibit(antagonists) or stimulate (agonists) T cell proliferation, leucocyteinfiltration, etc. Exemplary antibodies include polyclonal, monoclonal,humanized, bispecific and heteroconjugate antibodies.

[0227] a. Polyclonal Antibodies

[0228] Methods of preparing polyclonal antibodies are known to skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent, and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the PRO301, PRO362 orPRO245 polypeptide of the invention or a fusion protein thereof. It maybe useful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include but are not limited to keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examplesof adjuvants which may be employed include Freund's complete adjuvantand MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

[0229] b. Monoclonal Antibodies

[0230] Antibodies which recognize and bind to the polypeptides of theinvention or which act as antagonists thereto may, alternatively bemonoclonal antibodies. Monoclonal antibodies may be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, orother appropriate host animal., is typically immunized with animmunizing agent to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the immunizingagent. Alternatively, the lymphocytes may be immunized in vitro.

[0231] The immunizing agent will typically include the PRO301, PRO362 orPRO245 polypeptide of the invention, an antigenic fragment or a fusionprotein thereof. Generally, either peripheral blood lymphocytes (“PBLs”)are used if cells of human origin are desired, or spleen cells or lymphnode cells are used if non-human mammalian sources are desired. Thelymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

[0232] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, California and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0233] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst the polypeptide of the invention or having similar activity asthe polypeptide of the invention. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0234] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0235] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0236] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0237] The antibodies are preferably monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0238] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0239] c. Human and Humanized Antibodies

[0240] The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general., the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct Biol.,2:593-596 (1992)].

[0241] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and coworkers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0242] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., J. Immunol., 147(1):86-95 (1991); U.S. Pat. No.5,750,373]. Similarly, human antibodies can be made by introducing ofhuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

[0243] d. Bispecific Antibodies

[0244] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities may be for the polypeptide of the invention, the other oneis for any other antigen, and preferably for a cell-surface protein orreceptor or receptor subunit.

[0245] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 [1983]). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0246] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are cotransfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0247] e. Heteroconjugate Antibodies

[0248] Heteroconjugate antibodies are composed of two covalently joinedantibodies. Such antibodies have, for example, been proposed to targetimmune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and fortreatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It iscontemplated that the antibodies may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

[0249] f Effector Function Engineering

[0250] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance the effectiveness of theantibody in treating an immune related disease, for example. For examplecysteine residue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.176:1191-1195 (1992) and Shopes, B., J. Immunol. 148:2918-2922 (1992).Homodimeric antibodies with enhanced anti-tumor activity may also beprepared using heterobifunctional cross-linkers as described in Wolff etal. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody canbe engineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3:219-230 (1989).

[0251] g. Immunoconjugates

[0252] The invention also pertains to immunoconjugates comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, toxin (e.g. an enzymatically active toxin of bacterial, fungal,plant or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate).

[0253] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and¹⁸⁶Re.

[0254] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

[0255] In another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tisue pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) whichis conjugated to a cytotoxic agent (e.g. a radionucleotide).

[0256] h. Immunoliposomes

[0257] The proteins, antibodies, etc. disclosed herein may also beformulated as immunoliposomes. Liposomes containing the antibody areprepared by methods known in the art, such as described in Epstein etal., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556.

[0258] Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as doxorubicin) may be optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19)1484 (1989).

[0259] 10. Pharmaceutical Compositions

[0260] The active molecules of the invention, polypeptides andantibodies, as well as other molecules identified by the screeningassays disclosed above, can be administered for the treatment ofinflammatory diseases, in the form of pharmaceutical compositions.

[0261] Therapeutic formulations of the active molecule, preferably aPRO301, PRO362 or PRO245 polypeptide or antibody of the invention, areprepared for storage by mixing the active molecule having the desireddegree of purity with optional pharmaceutically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. [1980])), in the form of lyophilized formulationsor aqueous solutions. Acceptable carriers, excipients, or stabilizersare nontoxic to recipients at the dosages and concentrations employed,and include buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0262] Compounds identified by the screening assays of the presentinvention can be formulated in an analogous manner, using standardtechniques well known in the art.

[0263] Lipofections or liposomes can also be used to deliver thepolypeptide, antibody, or an antibody fragment, into cells. Whereantibody fragments are used, the smallest inhibitory fragment whichspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable region sequences of anantibody, peptide molecules can be designed which retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology (see, e.g.Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893 [1993]).

[0264] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine or growth inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

[0265] The active molecules may also be entrapped in microcapsulesprepared, for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0266] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0267] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g. films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37C, resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0268] 11. Methods of Treatment

[0269] It is contemplated that the polypeptides, antibodies and otheractive compounds of the present invention may be used to treat variousinflammatory diseases and conditions, such as T cell mediated diseases,including those characterized by infiltration of leucocyte cells into atissue, stimulation of T-cell proliferation, inhibition of T-cellproliferation, increased or decreased vascular permeability or theinhibition thereof.

[0270] The compounds of the invention (e.g., PRO301, PRO362, PRO245)encode new members of a family of proteins characterized by homology toA33 antigen. The proinflammatory nature of the compounds of theinvention is indicated in the in vitro assays below.

[0271] The proteins encoded by the DNA40628, DNA45416 and DNA35638compounds of the invention [(SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:9), respectively], share homology with identity with junctional adhesionmolecule (JAM), Martin-Padura et al., J. Cell Biol. 1998 142(1): 117-27.The most substantial identity is shared by the PRO301 protein encoded byDNA40628 (SEQ ID NO: 1) at 67%. JAM is involved in the recruitment ofmonocytes in response to MCP-1, MCP-3 and LPS in vivo. Antibodies to JAMblock monocyte transmigration in vivo. JAM is localized to the murineepithelia and endothelia as a junctional adhesion molecule for monocytetransmigration. Other leukocytes may also use JAM, but no informationsupports this notion. JAM is elevated in the colon of mice with colitisand likely plays a role in the recruitment of monocytes or leukocytesinto the colonic lesion.

[0272] Exemplary conditions or disorders to be treated with thepolypeptides, antibodies and other compounds of the invention, include,but are not limited to inflammatory bowel disease (i.e, ulcerativecolitis, Crohn's disease), systemic lupus erythematosis, rheumatoidarthritis, juvenile chronic arthritis, spondyloarthropathies, systemicsclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory and fibrotic lung diseases such as cystic fibrosis,gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease.

[0273] In systemic lupus erythematosus, the central mediator of diseaseis the production of auto-reactive antibodies to self proteins/tissuesand the subsequent generation of immune-mediated inflammation.Antibodies either directly or indirectly mediate tissue injury. Though Tlymphocytes have not been shown to be directly involved in tissuedamage, T lymphocytes are required for the development of auto-reactiveantibodies. The genesis of the disease is thus T lymphocyte dependent.Multiple organs and systems are affected clinically including kidney,lung, musculoskeletal system, mucocutaneous, eye, central nervoussystem, cardiovascular system, gastrointestinal tract, bone marrow andblood.

[0274] Rheumatoid arthritis (RA) is a chronic systemic autoimmuneinflammatory disease that mainly involves the synovial membrane ofmultiple joints with resultant injury to the articular cartilage. Thepathogenesis is T lymphocyte dependent and is associated with theproduction of rheumatoid factors, auto-antibodies directed against selfIgG, with the resultant formation of immune complexes that attain highlevels in joint fluid and blood. These complexes in the joint may inducethe marked infiltrate of lymphocytes and monocytes into the synovium andsubsequent marked synovial changes; the joint space/fluid if infiltratedby similar cells with the addition of numerous neutrophils. Tissuesaffected are primarily the joints, often in symmetrical pattern.However, extra-articular disease also occurs in two major forms. Oneform is the development of extra-articular lesions with ongoingprogressive joint disease and typical lesions of pulmonary fibrosis,vasculitis, and cutaneous ulcers. The second form of extra-articulardisease is the so called Felty's syndrome which occurs late in the RAdisease course, sometimes after joint disease has become quiescent, andinvolves the presence of neutropenia, thrombocytopenia and splenomegaly.This can be accompanied by vasculitis in multiple organs with formationsof infarcts, skin ulcers and gangrene. Patients often also developrheumatoid nodules in the subcutis tissue overlying affected joints; thenodules late stage have necrotic centers surrounded by a mixedinflammatory cell infiltrate. Other manifestations which can occur in RAinclude: pericarditis, pleuritis, coronary arteritis, intestitialpneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, andrhematoid nodules.

[0275] Juvenile chronic arthritis is a chronic idiopathic inflammatorydisease which begins often at less than 16 years of age. Its phenotypehas some similarities to RA; some patients which are rhematoid factorpositive are classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and istypically destructive and leads to joint ankylosis and retarded growth.Other manifestations can include chronic anterior uveitis and systemicamyloidosis.

[0276] Spondyloarthropathies are a group of disorders with some commonclinical features and the common association with the expression ofHLA-B27 gene product. The disorders include: ankylosing sponylitis,Reiter's syndrome (reactive arthritis), arthritis associated withinflammatory bowel disease, spondylitis associated with psoriasis,juvenile onset spondyloarthropathy and undifferentiatedspondyloarthropathy. Distinguishing features include sacroileitis withor without spondylitis; inflammatory asymmetric arthritis; associationwith HLA-B27 (a serologically defined allele of the HLA-B locus of class1 MHC); ocular inflammation, and absence of autoantibodies associatedwith other rheumatoid disease. The cell most implicated as key toinduction of the disease is the CD8+ T lymphocyte, a cell which targetsantigen presented by class 1 MHC molecules. CD8+ T cells may reactagainst the class 1 MHC allele HLA-B27 as if it were a foreign peptideexpressed by MHC class I molecules. It has been hypothesized that anepitope of HLA-B27 may mimic a bacterial or other microbial antigenicepitope and thus induce a CD8+ T cells response.

[0277] Systemic sclerosis (scleroderma) has an unknown etiology. Ahallmark of the disease is induration of the skin; likely this isinduced by an active inflammatory process. Scleroderma can be localizedor systemic; vascular lesions are common and endothelial cell injury inthe microvasculature is an early and important event in the developmentof systemic sclerosis; the vascular injury may be immune mediated. Animmunologic basis is implied by the presence of mononuclear cellinfiltrates in the cutaneous lesions and the presence of anti-nuclearantibodies in many patients. ICAM-1 is often upregulated on the cellsurface of fibroblasts in skin lesions suggesting that T cellinteraction with these cells may have a role in the pathogenesis of thedisease. Other organs involved include: the gastrointestinal tract:smooth muscle atrophy and fibrosis resulting in abnormalperistalsis/motility; kidney: concentric subendothelial intimalproliferation affecting small arcuate and interlobular arteries withresultant reduced renal cortical blood flow, results in proteinuria,azotemia and hypertension; skeletal muscle: atrophy, interstitialfibrosis; inflammation; lung: interstitial pneumonitis and interstitialfibrosis; and heart: contraction band necrosis, scarring/fibrosis.

[0278] Idiopathic inflammatory myopathies including dermatomyositis,polymyositis and others are disorders of chronic muscle inflammation ofunknown etiology resulting in muscle weakness. Muscleinjury/inflammation is often symmetric and progressive. Autoantibodiesare associated with most forms. These myositis-specific autoantibodiesare directed against and inhibit the function of components, proteinsand RNA's, involved in protein synthesis.

[0279] Sjögren's syndrome is due to immune-mediated inflammation andsubsequent functional destruction of the tear glands and salivaryglands. The disease can be associated with or accompanied byinflammatory connective tissue diseases. The disease is associated withautoantibody production against Ro and La antigens, both of which aresmall RNA-protein complexes. Lesions result in keratoconjunctivitissicca, xerostomia, with other manifestations or associations includingbilary cirrhosis, peripheral or sensory neuropathy, and palpablepurpura.

[0280] Systemic vasculitis are diseases in which the primary lesion isinflammation and subsequent damage to blood vessels which results inischemia/necrosis/degeneration to tissues supplied by the affectedvessels and eventual end-organ dysfunction in some cases. Vasculitidescan also occur as a secondary lesion or sequelae to otherimmune-inflammatory mediated diseases such as rheumatoid arthritis,systemic sclerosis, etc., particularly in diseases also associated withthe formation of immune complexes. Diseases in the primary systemicvasculitis group include: systemic necrotizing vasculitis: polyarteritisnodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener'sgranulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.Miscellaneous vasculitides include: mucocutaneous lymph node syndrome(MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease,thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizingvenulitis. The pathogenic mechanism of most of the types of vasculitislisted is believed to be primarily due to the deposition ofimmunoglobulin complexes in the vessel wall and subsequent induction ofan inflammatory response either via ADCC, complement activation, orboth.

[0281] Sarcoidosis is a condition of unknown etiology which ischaracterized by the presence of epithelioid granulomas in nearly anytissue in the body; involvement of the lung is most common. Thepathogenesis involves the persistence of activated macrophages andlymphoid cells at sites of the disease with subsequent chronic sequelaeresultant from the release of locally and systemically active productsreleased by these cell types.

[0282] Autoimmune hemolytic anemia including autoimmune hemolyticanemia, immune pancytopenia, and paroxysmal noctural hemoglobinuria is aresult of production of antibodies that react with antigens expressed onthe surface of red blood cells (and in some cases other blood cellsincluding platelets as well) and is a reflection of the removal of thoseantibody coated cells via complement mediated lysis and/orADCC/Fc-receptor-mediated mechanisms.

[0283] In autoimmune thrombocytopenia including thrombocytopenicpurpura, and immune-mediated thrombocytopenia in other clinicalsettings, platelet destruction/removal occurs as a result of eitherantibody or complement attaching to platelets and subsequent removal bycomplement lysis, ADCC or FC-receptor mediated mechanisms.

[0284] Thyroiditis including Grave's disease, Hashimoto's thyroiditis,juvenile lymphocytic thyroiditis, and atrophic thyroiditis, are theresult of an autoimmune response against thyroid antigens withproduction of antibodies that react with proteins present in and oftenspecific for the thyroid gland. Experimental models exist includingspontaneous models: rats (BUF and BB rats) and chickens (obese chickenstrain); inducible models: immunization of animals with eitherthyroglobulin, thyroid microsomal antigen (thyroid peroxidase).

[0285] Type I diabetes mellitus or insulin-dependent diabetes is theautoimmune destruction of pancreatic islet β cells; this destruction ismediated by auto-antibodies and auto-reactive T cells. Antibodies toinsulin or the insulin receptor can also produce the phenotype ofinsulin-non-responsiveness.

[0286] Immune mediated renal diseases, including glomerulonephritis andtubulointerstitial nephritis, are the result of antibody or T lymphocytemediated injury to renal tissue either directly as a result of theproduction of autoreactive antibodies or T cells against renal antigensor indirectly as a result of the deposition of antibodies and/or immunecomplexes in the kidney that are reactive against other, non-renalantigens. Thus other immune-mediated diseases that result in theformation of immune-complexes can also induce immune mediated renaldisease as an indirect sequelae. Both direct and indirect immunemechanisms result in inflammatory response that produces/induces lesiondevelopment in renal tissues with resultant organ function impairmentand in some cases progression to renal failure. Both humoral andcellular immune mechanisms can be involved in the pathogenesis oflesions.

[0287] Demyelinating diseases of the central and peripheral nervoussystems, including Multiple Sclerosis; idiopathic demyelinatingpolyneuropathy or Guillain-Barr syndrome; and Chronic InflammatoryDemyelinating Polyneuropathy, are believed to have an autoimmune basisand result in nerve demyelination as a result of damage caused tooligodendrocytes or to myelin directly. In MS there is evidence tosuggest that disease induction and progression is dependent on Tlymphocytes. Multiple Sclerosis is a demyelinating disease that is Tlymphocyte-dependent and has either a relapsing-remitting course or achronic progressive course. The etiology is unknown; however, viralinfections, genetic predisposition, environment, and autoimmunity allcontribute. Lesions contain infiltrates of predominantly T lymphocytemediated, microglial cells and infiltrating macrophages; CD4+Tlymphocytes are the predominant cell type at lesions. The mechanism ofoligodendrocyte cell death and subsequent demyelination is not known butis likely T lymphocyte driven.

[0288] Inflammatory and Fibrotic Lung Disease, including eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis may involve a disregulated immune-inflammatory response.Inhibition of that response would be of therapeutic benefit.

[0289] Autoimmune or Immune-mediated Skin Disease including Bullous SkinDiseases, Erythema Multiforme, and Contact Dermatitis are mediated byauto-antibodies, the genesis of which is T lymphocyte-dependent.

[0290] Psoriasis is a T lymphocyte-mediated inflammatory disease.Lesions contain infiltrates of T lymphocytes, macrophages and antigenprocessing cells, and some neutrophils.

[0291] Allergic diseases, including asthma; allergic rhinitis; atopicdermatitis; food hypersensitivity; and urticaria are T lymphocytedependent. These diseases are predominantly mediated by T lymphocyteinduced inflammation, IgE mediated-inflammation or a combination ofboth.

[0292] Transplantation associated diseases, including Graft rejectionand Graft-Versus-Host-Disease (GVHD) are T lymphocyte-dependent;inhibition of T lymphocyte function is ameliorative.

[0293] Other diseases in which intervention of the immune and/orinflammatory response have benefit are Infectious disease including butnot limited to viral infection (including but not limited to AIDS,hepatitis A, B, C, D, E) bacterial infection, fungal infections, andprotozoal and parasitic infections (molecules (or derivatives/agonists)which stimulate the MLR can be utilized therapeutically to enhance theimmune response to infectious agents), diseases of immunodeficiency(molecules/derivatives/agonists) which stimulate the MLR can be utilizedtherapeutically to enhance the immune response for conditions ofinherited, acquired, infectious induced (as in HIV infection), oriatrogenic (i.e. as from chemotherapy) immunodeficiency), and neoplasia.

[0294] It has been demonstrated that some human cancer patients developan antibody and/or T lymphocyte response to antigens on neoplasticcells. It has also been shown in animal models of neoplasia thatenhancement of the immune response can result in rejection or regressionof that particular neoplasm. Molecules that enhance the T lymphocyteresponse in the MLR have utility in vivo in enhancing the immuneresponse against neoplasia. Molecules which enhance the T lymphocyteproliferative response in the MLR (or small molecule agonists orantibodies that affected the same receptor in an agonistic fashion) canbe used therapeutically to treat cancer. Molecules that inhibit thelymphocyte response in the MLR also function in vivo during neoplasia tosuppress the immune response to a neoplasm; such molecules can either beexpressed by the neoplastic cells themselves or their expression can beinduced by the neoplasm in other cells. Antagonism of such inhibitorymolecules (either with antibody, small molecule antagonists or othermeans) enhances immune-mediated tumor rejection.

[0295] Additionally, inhibition of molecules with proinflammatoryproperties may have therapeutic benefit in reperfusion injury; stroke;myocardial infarction; atherosclerosis; acute lung injury; hemorrhagicshock; burn; sepsis/septic shock; acute tubular necrosis; endometriosis;degenerative joint disease and pancreatis.

[0296] The PRO301, 362 and PRO245 compounds of the present invention,e.g. polypeptides or antibodies, are administered to a mammal,preferably a human, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation (intranasal, intrapulmonary) routes. Intravenousor inhaled administration of polypeptides and antibodies is preferred.

[0297] In immunoadjuvant therapy, other therapeutic regimens, suchadministration of an anti-cancer agent, may be combined with theadministration of the proteins, antibodies or compounds of the instantinvention. For example, the patient to be treated with theimmunoadjuvants of the invention may also receive an anti-cancer agent(chemotherapeutic agent) or radiation therapy. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the immunoadjuvant or may be givensimultaneously therewith. Additionally, an anti-oestrogen compound suchas tamoxifen or an anti-progesterone such as onapristone (see, EP616812) may be given in dosages known for such molecules.

[0298] It may be desirable to also administer antibodies against otherimmune disease associated or tumor associated antigens, such asantibodies which bind to CD20, CD11a, CD 18, ErbB2, EGFR, ErbB3, ErbB4,or vascular endothelial factor (VEGF). Alternatively, or in addition,two or more antibodies binding the same or two or more differentantigens disclosed herein may be coadministered to the patient.Sometimes, it may be beneficial to also administer one or more cytokinesto the patient. In one embodiment, the polypeptides of the invention arecoadministered with a growth inhibitory agent. For example, the growthinhibitory agent may be administered first, followed by a polypeptide ofthe invention. However, simultaneous administration or administrationfirst is also contemplated. Suitable dosages for the growth inhibitoryagent are those presently used and may be lowered due to the combinedaction (synergy) of the growth inhibitory agent and the polypeptide ofthe invention.

[0299] For the treatment or reduction in the severity of immune relateddisease, the appropriate dosage of an a compound of the invention willdepend on the type of disease to be treated, as defined above, theseverity and course of the disease, whether the agent is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the compound, and the discretion of theattending physician. The compound is suitably administered to thepatient at one time or over a series of treatments. Preferably, it isdesireable to determine the dose-response curve and the pharmaceuticalcomposition of the invention first in vitro, and then in useful animalmodels prior to testing in humans.

[0300] For example, depending on the type and severity of the disease,about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of polypeptide or antibodyis an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. A typical daily dosage might range from about 1ug/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

[0301] 12. Articles of Manufacture

[0302] In another embodiment of the invention, an article of manufacturecontaining materials useful for the diagnosis or treatment of thedisorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for diagnosing ortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The active agentin the composition is usually a polypeptide or an antibody of theinvention. The label on, or associated with, the container indicatesthat the composition is used for diagnosing or treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

[0303] 13. Diagnosis and Prognosis of Immune Related Disease

[0304] Cell surface proteins, such as proteins which are overexpressedin certain immune related diseases, are excellent targets for drugcandidates or disease treatment. The same proteins along with secretedproteins encoded by the genes amplified in immune related disease statesfind additional use in the diagnosis and prognosis of these diseases.For example, antibodies directed against the protein products of genesamplified in multiple sclerosis, rheumatoid arthritis, or another immunerelated disease, can be used as diagnostics or prognostics.

[0305] For example, antibodies, including antibody fragments, can beused to qualitatively or quantitatively detect the expression ofproteins encoded by amplified or overexpressed genes (“marker geneproducts”). The antibody preferably is equipped with a detectable, e.g.fluorescent label, and binding can be monitored by light microscopy,flow cytometry, fluorimetry, or other techniques known in the art. Thesetechniques are particularly suitable, if the overexpressed gene encodesa cell surface protein Such binding assays are performed essentially asdecribed above.

[0306] In situ detection of antibody binding to the marker gene productscan be performed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat-a wide variety of histological methods are readily available for insitu detection.

[0307] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0308] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES

[0309] Commercially available reagents referred to in the examples wereused according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Rockville, Md.

Example 1 Isolation of cDNA clones Encoding Human PRO301

[0310] The extracellular domain (ECD) sequences (including the secretionsignal sequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public EST databases (e.g., GenBank), a proprietaryEST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif. ). Thesearch was performed using the computer program BLAST or BLAST2[Altschul et al., Methods in Enzymology, 266:460-480 (1996);http://blast.wustl/edu/blast/README.html] as a comparison of the ECDprotein sequences to a 6 frame translation of the EST sequences. Thosecomparisons resulting in a BLAST score of 70 (or in some cases, 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.;http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).

[0311] A consensus DNA sequence encoding DNA35936 was assembled usingphrap. In some cases, the consensus DNA sequence was extended usingrepeated cycles of blast and phrap to extend the consensus sequence asfar as possible using the three sources of EST sequences listed above.

[0312] Based on this consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence. Forward and reverse PCR primers (notated as*.f and *.r, respectively) may range from 20 to 30 nucleotides(typically about 24), and are designed to give a PCR product of 100-1000bp in length. The probe sequences (notated as *.p) are typically 40-55bp (typically about 50) in length. In some cases, additionaloligonucleotides are synthesized when the consensus sequence is greaterthan 1-1.5 kbp. In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification, as per Ausubel et al., Current Protocols in MolecularBiology, with the PCR primer pair. A positive library was then used toisolate clones encoding the gene of interest by the in vivo cloningprocedure suing the probe oligonucleotide and one of the PCR primers.

[0313] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with the PCR primer pair identified above. A positivelibrary was then used to isolate clones encoding the PRO301 gene usingthe probe oligonucleotide and one of the PCR primers.

[0314] RNA for construction of the cDNA libraries was isolated fromhuman fetal kidney. The cDNA libraries used to isolated the cDNA cloneswere constructed by standard methods using commercially availablereagents (e.g., Invitrogen, San Diego, Calif.; Clontech, etc.) The cDNAwas primed with oligo dT containing a NotI site, linked with blunt toSalI hemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

[0315] A cDNA clone was sequenced in its entirety. The full lengthnucleotide sequence of native sequence DNA40628 is shown in FIG. 5 (SEQID NO: 11). Clone DNA40628 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 52-54(FIG. 5; SEQ ID NO: 11). The predicted polypeptide precursor is 299amino acids long with a predicted molecular weight of 32583 daltons andpI of 8.29. Clone DNA40628 has been deposited with ATCC and is assignedATCC deposit No. 209432.

[0316] Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence, PRO301 encoded by DNA40628 shows amino acidsequence identity to A33 antigen precursor (30%) and coxsackie andadenovirus receptor protein (29%).

[0317] The oligonucleotide sequences used in the above procedure werethe following: OLI2162 (35936.f1)(SEQ ID NO: 12)TCGCGGAGCTGTGTTCTGTTTCCC OLI2163 (35936.p1)(SEQ ID NO: 13)TGATCGCGATGGGGACAAAGGCGCAAGCTCGAGAGGAAACTGTTGTGCCT OLI2164(35936.f2)(SEQ ID NO: 14) ACACCTGGTTCAAAGATGGG OLI2165 (35936.r1)(SEQ IDNO: 15) TAGGAAGAGTTGCTGAAGGCACGG OLI2166 (35936.f3)(SEQ ID NO: 16)TTGCCTTACTCAGGTGCTAC OLI2167 (35936.r2)(SEQ ID NO: 17)ACTCAGCAGTGGTAGGAAAG

Example 2 Isolation of cDNA Clones Encoding Human PRO362

[0318] The extracellular domain (ECD) sequences (including the secretionsignal, if any) of about 950 known secreted proteins from the Swiss-Protpublic protein database were used to search expressed sequences tag(EST) databases. The EST databases included public EST databases (e.g.,GenBank) and a proprietary EST DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.). The search was performed using thecomputer program BLAST or BLAST-2 (e.g., Altshul et al., Methods inEnzymology 266: 460-480 (1996)) as a comparison of the ECD proteinsequences to a 6 frame translation of the EST sequence. Thosecomparisons resulting in a BLAST score 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.

[0319] A consensus DNA sequence was assembled relative to other ESTsequences using phrap. This consensus sequence is herein designatedDNA42257 (SEQ ID NO: 5) (see FIG. 4C). Based on the DNA42257 (SEQ ID NO:5) consensus sequence shown in FIG. 4C, oligonucleotides weresythesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO362. Forward and reverse PCR primersgenerally range from 20 to 30 mucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

[0320] PCR primers (forward and reverse) were synthesized: forward PCRprimer 1 (42257.f1) 5′-TATCCCTCCAATTGAGCACCCTGG-3′ (SEQ ID NO: 18)forward PCR primer 2 (42257.f2) 5′-GTCGGAAGACATCCCAACAAG-3′ (SEQ ID NO:19) reverse PCR primer 1 (42257.r1) 5′-CTTCACAATGTCGCTGTGCTGCTC-3′ (SEQID NO: 20) reverse PCR primer 2 (42257.r2 5′-AGCCAAATCCAGCAGCTGGCTTAC-3′(SEQ ID NO: 21)

[0321] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consenus DNA42257 sequence which had the followingnucleotide sequence:

[0322] Hybridization probe (42257.p1)

[0323] 5′-TGGATGACCGGAGCCACTACACGTGTGAAGTCACCTGGCAGACTCCTGAT-3′ (SEQ IDNO: 22).

[0324] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with the PCR primer pairs identified above. A positivelibrary was then used to isolate clones encoding the PRO362 gene usingthe probe oligonucleotide and one of the PCR primers.

[0325] RNA for construction of the cDNA libraries was isolated fromhuman fetal brain tissue (LIB 153). The cDNA libraries used to isolatethe cDNA clones were constructed by standard methods using commerciallyavailable reagents such as those from Invitrogen, San Diego, Calif. ThecDNA was primed with oligo dT containing a NotI site linked with bluntto SalI hemikinased adaptors, cleaved with NotI, sized appropriately begel electrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRK5D thatdoes not contain the SfiI site; see Holmes et al., Science 253:1278-1280 (1991)) in the unique XhoI and NotI sites.

[0326] DNA sequencing of the clones isolated as described gave thefull-length DNA sequence for an isolated PRO362 [herein designated asUNQ317 (DNA45416-1251)(SEQ ID NO: 7).

[0327] The entire nucleotide sequence of UNQ317 (DNA45416-1251) is shownin FIG. 6 (SEQ ID NO: 7). Clone UNQ367 (DNA45416-1251) (SEQ ID NO: 7)contains a single open reading frame with an apparent translationalinitiation site at nucleotide positions 1082-1084 (FIG. 6, SEQ ID NO:7). The predicted polypeptide precursor is 321 amino acids long (FIG. 3,SEQ ID NO: 2). The full-length PRO362 protein shown if FIG. 3 has anestimated molecular weight of about 35,544 daltons and a pI of about8.51. Analysis of the full-length PRO362 polypeptide as shown in FIG. 3(SEQ ID NO: 2) evidences the presence of a glycosaminoglycan attachmentsite at about amino acid 149 to about amino acid 152 and a transmembranedomain from about amino acid 276 to about amino acid 306. Clone UTNQ317(DNA45416-1251) has been deposited with ATCC desposit No.: 209620.

Example 3 Isolation of cDNA Clones Encoding Human PRO245

[0328] The extracellular domain (ECD) sequences (including the secretionsignal., if any) of about 950 known secreted proteins from theSwiss-Prot public protein database were used to search expressedsequences tag (EST) databases. The EST databases included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.). The search was performedusing the computer program BLAST or BLAST-2 (e.g., Altshul et al.,Methods in Enzynology 266: 460-480 (1996)) as a comparison of the ECDprotein sequences to a 6 frame translation of the EST sequence. Thosecomparisons resulting in a BLAST score 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.

[0329] A consensus DNA sequence was assembled relative to other ESTsequences, wherein the consensus sequence is herein designated DNA30954(SEQ ID NO: 27). Based on the DNA30954 consensus sequence,oligonucleotides were synthesized to identify by PCR a cDNA library thatcontained the sequence of interest and for use as probes to isolate aclone of the full-length coding sequence for PRO245.

[0330] A pair of PCR primers (forward and reverse) were synthesized:forward PCR 5′-ATCGTTGTGAAGTTAGTGCCCC-3′ (SEQ ID primer NO: 28) reversePCR 5′-ACCTGCGATATCCAACAGAATTG-3′ (SEQ ID primer NO: 29)

[0331] Forward and reverse PCR primers generally range from 20 to 30mucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair.

[0332] Additionally, a synthetic oligonucleotide hybridization probeswas constructed from the consensus DNA30954 sequences which had thefollowing nucleotide sequence:

[0333] hybridization probe:

[0334] 5′-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGTTCC-3′ (SEQ IDNO: 30)

[0335] In order to screen several libraries for a source of afull-length clone, DNA form the libraries was screened by PCRamplification with the PCR primer pair identified above. A positivelibrary was then used to isolated clones encoding the PRO245 gene usingthe probe oligonucleotide and one of the PCR primers.

[0336] RNA for construction of the cDNA libraries was isolated fromhuman fetal liver tissue. The cDNA libraries used to isolate the cDNAclones were constructed by standrd methods using commercially availablereagents such as those from Invitrogen, San Diego, CA. The cDNA wasprimed with oligo cT containing a notI site, linked with blunt to SalIhemokinased adaptors, cleaved with Noti, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRKSD thatdoes not contain the SfiI site; see Holmes et al., Science 253:1278-1280 (1991)) in the unique XhoI and NotI sites.

[0337] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a native sequence PRO245 [herein designatedas UNQ219 (DNA35638)(SEQ ID NO: 8)] and the derived protein sequence(SEQ ID NO: 9).

[0338] The entire nucleotide sequence of UNQ219 (DNA35638) is shown inFIG. 7 (SEQ ID NO: 8). Clone UNQ219 (DNA35638)(SEQ ID NO: 8) contains asingle open reading frame with an apparent translational initiation siteat nucleotide positions 89-91 [Kozak et al., supra] and ending at thestop codon at nucleotide positions 1025-1027 (FIG. 7, SEQ ID NO: 8). Thepredicted polypeptide precursor is 312 amino acids long (FIG. 11)(SEQ IDNO: 9). Clone UNQ219 (DNA35638) has been deposited with the ATCC on Sep.17, 1997 and is assigned ATCC deposit No. 209265.

Example 4 Inhibition of VEGF Stimulated Proliferation of EndothelialCell Growth

[0339] Bovine adrenal cortical capillary endothelial (ACE) cells (fromprimary culture, maximum 12-14 passages) were plated on 96-wellmicrotiter plates (Amersham Life Science) at a density of 500 cells/wellper 100 μL in low glucose DMEM, 10% calf serum, 2 mM glutamine, 1×pen/strept and fungizone, supplemented with 3 ng/mL VEGF. Controls wereplated the same way but some did not include VEGF. A test sample of thePRO301 and PRO245 polypeptide was added in a 100 μl volume for a 200 mcLfinal volume. Cells were incubated for 6-7 days at 37° C. The media wasaspirated and the cells washed lx with PBS. An acid phosphatase reactionmixture (100 μL, 0.1M sodium acetate, pH 5.5, 0.1% Triton-100, 10 mMp-nitrophenyl phosphate) was added. After incubation for 2 hours at 37°C., the reaction was stopped by addition of 10 mcL 1N NaOH. OD wasmeasured on microtiter plate reader at 405 nm. Controls were no cells,cells alone, cells+FGF (5 ng/mL), cells+VEGF (3 ng/mL), cells+VEGF (3ng/ml)+TGF-β (1 ng/ml), and cells+VEGF (3ng/mL)+LIF (5 ng/mL). (TGF-β ata 1 ng/ml concentration is known to block 70-90% of VEGF stimulated cellproliferation.)

[0340] The results were assessed by calculating the percentageinhibition of VEGF (3 ng/ml) stimulated cells proliferation, determinedby measuring acid phosphatase activity at OD₄₀₅ nm, (1) relative tocells without stimulation, and (2) relative to the reference TGF-βinhibition of VEGF stimulated activity. The results, shown in Table 1,are indicative of the utility of the PRO301 and PRO245 polypeptide inthe inhibition of cell growth, especially cancer therapy andspecifically in inhibiting tumor angiogenesis. TABLE 1 % ProliferationCompound Tested Concentation relative to control DNA40628 protein (SEQID NO: 1)  7.0 nM 1.02 DNA40628 protein (SEQ ID NO: 1)  70.0 nM 0.88DNA40628 protein (SEQ ID NO: 1) 700.0 nM 0.44 DNA40628 protein (SEQ IDNO: 1) 0.01% 0.92 DNA40628 protein (SEQ ID NO: 1)  0.1% 0.85 DNA40628protein (SEQ ID NO: 1)  1.0% 0.68 DNA35638 protein (SEQ ID NO: 9) 0.01%0.76 DNA35638 protein (SEQ ID NO: 9)  0.1% 0.35 DNA35638 protein (SEQ IDNO: 9)  1.0% 0.11 DNA35638 protein (SEQ ID NO: 9)  0.48 nM 1.03 DNA35638protein (SEQ ID NO: 9)  4.8 nM 0.95 DNA35638 protein (SEQ ID NO: 9) 48.0 nM 0.49

Example 5 Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay

[0341] This example shows that the polypeptides of the invention areactive as a stimulator of the proliferation of stimulated T-lymphocytes.Compounds which stimulate proliferation of lymphocytres are usefultherapeutically where enhancement of an inflammatory response isbeneficial. Compounds which inhibit proliferation of lymphocytes areuseful therapeutically where suppression of inflammatory response. Atherapeutic agent may take the form of antagonists of the polypeptide ofthe invention, for example, murine-human chimeric, humanized or humanantibodies against the polypeptide.

[0342] The basic protocol for this assay is described in CurrentProtocol in Immunology, Unit 3.12, J. E. Coligan, A. M. Kruisbeek, D HMarglies, E M Shevach and W Strober, Eds, National Institute of Health,Published by John Wiley & Sons, Inc.

[0343] More specifically, in one assay variant, peripheral bloodmononuclear cells (PBMC) are isolated from mammalian individuals, forexample a human volunteer, by leukopheresis (one done will supplystimulatory PBMCs, the other donor will supply responder PBMCs). Ifdesired, the cells are frozen in fetal bovine serum and DMSO afterisolation. Frozen cells may be thawed overnight in assay media (37° C.,5% CO₂) and then washed and resuspended to 3×106⁶ cells/ml of assaymedia (RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1%glutamine, 1% HEPES, 1% non-essential amino acids, 1% pyruvate).

[0344] The stimulator of PBMCs are prepared by irradiating the cells(about 3000 Rads). The assay is prepared by plating in triplicate wellsa mixture of: 100 μl of test sample diluted to 1% of 0.1%; 50 μl ofirradiated stimulator cells and 50 μl of responder PBMC cells. 100 μL ofcell culture media or 100 ml of CD4-IgG is used as the control. Thewells are then incubated at 37° C., 5% CO₂ for 4 days. On day 5, eachwell is pulsed with tritiated thymidine (1.0 mC/well; Amersham). Afterhours the cells are washed 3 times and then the uptake of the label isevaluated.

[0345] In another variant of this assay, PBMC's are isolated from thespleens of Balb/c mice and C57B6 mice. The cells are teased from freshlyharvested spleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting ans washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of asssay media. Theassay is then conducted as described above. The results of this assayfor compounds of the invention are shown below in Table 2. Positiveincreases over control are considered positive with increases of greaterthan or equal to 180% being preferred. However, any value greater thancontrol indicates a stimulatory effect for the test protein. TABLE 2Percent Increase Compound Concentration over Control DNA40628 protein(SEQ ID NO: 1) 0.1% 181.7 DNA40628 protein (SEQ ID NO: 1) 1.0% 187.3DNA40628 protein (SEQ ID NO: 1) 0.1% 193.4 DNA40628 protein (SEQ IDNO: 1) 1.0% 204.1 DNA45416 protein (SEQ ID NO: 2) 0.1% 87.4 DNA45416protein (SEQ ID NO: 2) 1.0% 180.2 DNA35638 protein (SEQ ID NO: 9) 0.1%189.7 DNA35638 protein (SEQ ID NO: 9) 0.1% 193.7 DNA35638 protein (SEQID NO: 9) 1.0% 212.5 DNA35638 protein (SEQ ID NO: 9) 1.0% 300.5

Example 6 Inflammatory Cell Infiltrates into Guinea Pig Skin

[0346] The following example shows that the polypeptides of theinvention are proinflammatory in that they stimulate inflammatory cellinfiltrates (i.e., neutrophilic, eosinophilic, monocytic or lymphocytic)into guinea pig skin. The assay described herein monitors the capacityof each protein to induce an inflammatory cell infiltrate into the skinof a guinea pig. Compounds which stimulate inflammatory infiltration areuseful therapeutically were enhancement of an inflammatory response isbeneficial. Compounds which inhibit proliferation of lymphocytes areuseful therapeutically where suppression of an inflammatory response isbeneficial. A therapeutic agent may take the form of antagonists of thepolypeptides of the invention, for example, murine-human chimeric,humanized or human antibodies against the polypeptide.

[0347] Hairless guinea pigs (Charles River Labs) weighing 350 grams ormore are anesthetized with ketamine (75-80 mg/kg body weight) andXylazine (5 mg/kg body weight) intramuscularly. The protein samples areinjected intradermally onto the backs of each animal at a volume of 100μl per injection site. There are approximately 16-24 injection sites peranimal. One mL of Evans blue dye (1% in physiological buffered saline)is injected intracardially. The animals are euthanized after 6 hours.Each skin injection site is biopsied and fixed in formalin. The skinsare prepared for histopathological evaluation. Each site is evaluatedfor inflammatory cell infiltration into the skin. Sites with visibleinflammatory cells are scored as positive. Samples inducing aninflammatory cell infiltrate are scored as proinflammatory substances.TABLE 3 Compound Proinflammatory activity DNA40628 protein (SEQ IDNO: 1) + DNA45416 protein (SEQ ID NO: 2) + DNA35638 protein (SEQ ID NO:9) + Negative control −

Example 7 Interaction with Human Neutrophils

[0348] The following example shows the ability of the polypeptides ofthe invention to bind to human neutrophils, a molecule associated withinflammation and the inflammatory response.

[0349] Neutrophils isolated from the blood of human donors (PMN) asdescribed in Scan. J. Clin. Lab Invest. Suppl. 97: 51-76 (1968), wereincubated with an Ig-fusion of protein encoded by DNA40628 (prepared asdiscussed in the following examples) or a negative control humanizedantibody.

[0350] The PMNs were resuspended in a microfuge tube in PBS at a densityof 2×10⁶ cell equivalents per condition. The cells were washed twicewith ice cold PBS and pelleted at 400×g between washes. The PMN cellswere blocked with 0.5% BSA in PBS (blocking reagent) at 4° C. for 1hour. After the incubation, the cells were further washed two additionaltimes with blocking reagent. The PMNs were pelleted after the final washand resuspended in 1 ml of blocking buffer at 0.1 μg/ml in both DNA40628protein and control antibody. The incubation was carried out for 2 hoursat 4° C. The PMN cells were gently resuspended every 15 minutes on ice,then washed and pelleted 5 times in blocking buffer, with each washlasting 5 minutes at 4° C. and pelleting occurring at 400×g. A 1:1000dilution of goat and anti-human IgG Fc specific-alkalinephosphatase-conjugated in the blocking buffer was then applied to thePMN cells. The PMN cells were incubated for 1 hour at 4° C., with gentlymixing every 15 minutes on ice. The PMN cells were then washed 5 timeswith blocking buffer, resuspended in the appropriate substrate foralkaline phosphatase and distributed in 4 equi-100 μl aliquots onto amicrotiter plate. Color development was read at O.D. 405. The resultsare shown in FIG. 21.

Example 8 Dot Blot Tissue Hybridization

[0351] A human RNA master blot (Clontech) was hybridized overnight at65° C. in Expresshyb® buffer (Clontech) per the manufacturer'sinstructions with 100 nM of psoralen-biotin labeled DNA40628 cDNA probe(SEQ ID NO: 7). Streptavidin-alkaline phosphatase was used to detect thebiotinylated probe. The blot was developed with CDP-star substrate(Ambion) and exposed for various times on Biomax film (Kodak). A cDNAhybridization analysis of human tissues show that DNA40628 mRNA isexpressed in many tissues except the cerebellum and spinal cord FIG. 19.DNA40628 mRNA is highly expressed in the colon, prostate, stomach,ovary, salivary gland, kidney, lung, trachea and placenta.

Example 9 Gene Product Overexpression

[0352] This example shows that genes encoding various proteins indicatedin FIG. 20 are overexpressed in colitic colon of CRF2-4−/− “knock out”mice. Therapeutic agents may take the form of antagonists of theindicated gene products, for example, murine-human chimeric, humanizedor human antibodies thereagainst.

[0353] CRF 2-4−/− mice (Spencer et al., J. Exp. Med. 187, 571-578(1998)), are animals which have a subunit of the gene encoding the IL-10receptor removed. The mice are unresponsive to the downregulatoryfunctions of IL-10 for macrophage activation, and cannot downregulateresponse to lipopolysaccharide triggering of macrophage TNF-α secretion.They develop a chronic colitis which can lead to colonic adenocarcinoma.

[0354] The probes for the proteins indicated in FIG. 20 were createdfrom mRNA templates for the indicated gene products and used in the5′-nuclease assay (e.g., TaqMan™) and real-time quantitative PCR (e.g.,ABI Prizm 7700 Sequence Detection System™ (Perkin-Elmer, AppliedBiosystems Division, Foster City, Calif.). The results are resported indelta CT units. One unit corresponds to 1 PCR cycle or approximately a2-fold amplification relative to normal, two units correspond to 4-fold,3 units to 8-fold, etc. Quantitation was obtained using primers and aTaqMan™ fluorescent tagged-mRNA derived from the testedinflammatory-related gene products indicated in FIG. 20. Regions of theindicated gene products which are most likely to contain unique nucleicacid sequences and which are least likely to have spliced out intronsare preferred for the primer derivation, e.g. 3′-untranslated region.

[0355] The 5′-nuclease assay reaction is a fluorescent PCR-basedtechnique which makes use of the 5′-exonuclease activity of Taq DNApolymerase enzyme to monitor amplification in real time. Twooligonucleotide primers are used to generate an amplicon typical of aPCR reaction. A third oligonucleotide, or probe, is designed to detectnucleotide sequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areported fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the probe is cleaved bythe Taq DNA polymerase enzyme in a template-dependent manner. Theresultant probe fragments disassociate in solution, and the signal fromthe release reporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmoleucle synthesized, and detection of the unquenched reporter dyeprovided the basis for quantitative interpretation of the data.

[0356] The 5′-nuclease procedure is run on a real-time quantiative PCRdevice such as the ABI Prism ₇₇₀₀™ Sequence Detection. The systemconsists of a thermocycler, laser, charge-coupled device (CCD) cameraand computer. The system amplifies samples in a 96-well format on athermocycler. During amplification, laser-induced fluorescent signal iscollected in real-time through fiber optics cables for all 96 wells, anddetected at the CCD. The system includes software for running theinstrument and for analyzing the data.

[0357] The 5′-nuclease assay data are initially expressed as Ct, or thethreshold cycle. This is defined as the cycle at which the reportersignal accumulates above the background level of fluorescence. The Ctvalues are used as quantitative measurement of the relative number ofstarting copies of a particular target sequence in a nucleic acidsample.

[0358] The results of the mRNA amplification are shown in FIG. 20.Expression in wild-type animals were compared with CRF2.4 KO animalswith beta-actin as the reference standard. Four animals were measured ineach group. All four KO animals were diagnosed with colitis and inaddition, three of these had colon adenocarcinoma.

[0359]FIG. 18 shows that JAM mRNA is increased 3.3-fold in the colon ofCRF2-4−/− mice with colitis. These mice are IL-10 receptor knock outsthat develop a spontaneous colitis mediated by lymphocytes, monocytesand neutrophils. IL-10 suppresses the inflammatory response bymodulating expression of certain inflammatory cytokines.

[0360] As a result, it is likely that PRO301, PRO362 and PRO245 wouldalso have elevated expression in inflammatory human disease, such asinflammatory bowel disease and other inflammatory diseases of the gut.

Example 10 Induction of Endothelial Cell Apoptosis

[0361] The ability of the polypeptides of the invention to induceapoptosis in endothelial cells was tested in human venous umbilical veinendothelial cells (HUVEC, Cell Systems). The first day, the cells wereplated on 96-well microtiter plates (Amersham Life Sciences, cytostar-Tscintillating microplate, RPNQ160, sterile, tissue-culture treated,individually wrapped), in 10% serum (CSG-medium, Cell Systems), at adensity of 2×10⁴ cells per well in a total volume of 100 μl. The secondday, PRO301 and PRO245 polypeptide encoded by DNA40628 and DNA35638,respectively, was added in triplicate at dilutions of 1%, 0.33% and0.11%. On the third day, the ability of the PRO301 and PRO245polypeptides to induce apoptosis was determined using a commericallyavailable kit, Apoptosis Detection Kit (R&D Systems, Minnesota) in whichannexin V, a member of the calcium and phospholipid binding proteins, isused to detect apoptosis, following the protocol recommended by themanufacturer. Fluroescein-labeled annexin V and propidium iodide wereadded to the cells. Analysis was performed with cytometers equipped witha single laser emitting excitation light at 488 nm. In this test, livecells will not stain with either fluorochrome, necrotic cells will stainwith both fluorochromes, and cells undergoing apoptosis will stain onlywith the annexin V-FITC reagent. The annexin V-FITC generated signal wasdetected in the FITC signal detector. The results are indicated in theTable 4 below. TABLE 4 % over background Compound tested Concentrationfluorescence DNA40628 protein (SEQ ID NO: 1) 0.11% 115.8 DNA40628protein (SEQ ID NO: 1) 0.33% 199.3 DNA40628 protein (SEQ ID NO: 1)  1.0%335.6 DNA35638 protein (SEQ ID NO: 9) 0.11% 77.6 DNA35638 protein (SEQID NO: 9) 0.33% 143.7 DNA35638 protein (SEQ ID NO: 9)  1.0% 146.0DNA35638 protein (SEQ ID NO: 9)  6.82 nM 67.2 DNA35638 protein (SEQ IDNO: 9) 20.46 nM 102.6 DNA35638 protein (SEQ ID NO: 9)  62.0 nM 118.8

[0362] The ability of the protein compounds of the invention to induceendothelial cell apoptosis , particularly in combination with thedisruption of cell junction formation as indicated in Example 4 isindicative that the compounds play roles in cell adhesion andtransmigration. Similar to murine JAM, the compounds are likely celljunction molecules in epithelia and endothelia, which explains theirbroad tissue distribution. The disruption of the induction ofendothelial cell apoptosis supports a role in cell growth and apoptosis.

Example 11 In Vitro Antitumor Assay

[0363] The antiproliferative activity of the PRO301 and PRO362polypeptides of the invention was determined in the investigational.,disease-orientated in vitro anti-cancer drug discovery assay of theNational Cancer Institute (NCI), using sulforhodamine B (SRB) dyebinding assay essentially as described by Skehan et al., J. Natl. CancerInst. 82: 1107-1112 (1990). The 60 tumor cell lines employed in thisstudy (“the NCI panel”) as well as conditions for their maintenance andculture in vitro have been described by Monks et al., J. Natl. CancerInst. 83: 757-766 (1991). The purpose of this screen is to initiallyevaluate the cytotoxic and/or cytostatic activity of the test compoundsagainst different types of tumors (Monks et al., supra, Boyd, Cancer:Princ. Pract. Oncol. Update 3(10): 1-12 (1989)).

[0364] Cell from approximately 60 human tumor cell lines were harvestedwith trypsin/EDTA (Gibco), washed once, resuspended in IMEM and theirviability was determined. The cell suspensions were added by pipet (100μL volume) into separate 96-well microtiter plates. The cell density forthe 60-day incubation was less than for the 20 day incubation to preventovergrowth. Inoculates were allowed a preincubation period of 24 hoursat 37° C. for stabilization. Dilutions at twice the intended testconcentration were added at time zero in 100 ml aliquots to themicrotiter plates wells (1:2 dilution). Test compounds were evaluated atgive half-log dilutions (1000 to 100,000 fold). Incubations took placefor two days and six days in a 5% CO₂ atmosphere and 100% humidity.

[0365] After incubation, the medium was removed and the cells were fixedin 0.1 ml of 10% trichloroacetic acid at 40° C. The plates were rinsedfive times with deionized water, dried, stained for 30 minutes with 0.1ml of 0.4% sulforhodamine B dye (Sigma) dissolved in 1% acetic acid,rinsed four times with 1% acetic acid to remove unbound dye, dried, andthe stain was extracted for five minutes with 0.1 ml of 10 mM Tris base[tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) ofsulforhodamine B at 492 nm was measured using a computer-interfaced,96-well microtiter plate reader.

[0366] A test sample is considered positive if it shows at least 50%growth inhibitory effect at one or more concentrations. The results areshown in the following tables, where the abbreviations are as follows:

[0367] NSCL=non-small lung carcinoma

[0368] CNS=central nervous system

[0369] Leuk=leukemia TABLE 5 Length of Tumor cell line Test compoundConcentration assay Type Designation DNA40628 protein 0.075 nM 6 ColonHCC-2998 (SEQ ID NO: 1) Melanoma M14 DNA40638 protein   700 nM 6Melanoma M14 (SEQ ID NO: 1) DNA40628 protein   152 nM 6 Colon SR (SEQ IDNO: 1) Melanoma LOX IMVI DNA40628 protein  15.2 nM 6 Melanoma LOX IMVI(SEQ ID NO: 1) DNA40628 protein  0.85 nM 6 NSCL HOP62 (SEQ ID NO: 1)Ovarian OVCAR-3 Prostate PC3 DNA45416 protein   15 nM 2 Ovarian SK-OV-3(SEQ ID NO: 2) DNA45416 protein   15 nM 6 NSCL NCI-H322M (SEQ ID NO: 2)Prostate PC-3 DNA45416 protein  4.7 nM 6 Melanoma LOX IMVI (SEQ ID NO:2) DNA45416 protein   47 nM 6 NSCL NCI-H322M (SEQ ID NO: 2) Colon Colo205 DNA45416 protein   152 nM 2 CNS SR-295 (SEQ ID NO: 2) Breast T047DDNA45416 protein   152 nM 6 Leuk SR, HL-60 (TB), MOLT-4, (SEQ ID NO: 2)K-562 NSCL NCI-H23, EKVX Colon HCC-2998 CNS U251 Melanoma UACC-62,UACC-257, LOX LMVI DNA35638 protein  0.35 nM 2 NSCL HOP92 (SEQ ID NO: 9)Ovarian OVCAR-4 DNA35638 protein  0.35 nM 2 Leuk SR (SEQ ID NO: 9)DNA35638 protein  0.35 nM 6 Colon HCC-2998 (SEQ ID NO: 9) DNA35638protein  3.5 nM 6 Leuk SR (SEQ ID NO: 9) Colon SW-620 DNA35638 protein 6.2 nM 6 Colon HCT-116 (SEQ ID NO: 9) DNA35638 protein  6.2 nM 6 LeukRPMI-8226 (SEQ ID NO: 9)

Example 12 Use of PRO301, PRO362 or PRO245 as a Hybridization Probe

[0370] The following method describes use of a nucleotide sequenceencoding a PRO301, PRO362 or PRO245 as a hybridization probe.

[0371] DNA comprising the coding sequence of native sequence PRO301,PRO362 or PRO245 (as shown in FIGS. 5-7, SEQ ID NO: 11, 7 and 8),respectively, is employed as a probe to screen for homologous DNAs (suchas those encoding naturally-occurring variants of PRO301, PRO362 orPRO245, respectively) in human tissue cDNA libraries or human tissuegenomic libraries.

[0372] Hybridization and washing of filters containing either libraryDNAs is performed under the following high stringency conditions.Hybridization of radiolabeled PRO301-, PRO362- or PRO245-derived probeto the filters is performed in a solution of 50% formamide, 5×SSC, 0.1%SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8,2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours.Washing of the filters is performed in an aqueous solution of 0.1×SSCand 0.1% SDS at 42° C.

[0373] DNAs having a desired sequence identity with the DNA encoding afull-length native sequence PRO301, PRO362 or PRO245 can then beidentified using standard techniques known in the art.

Example 13 Expression of PRO301, PRO362 or PRO245 in E. coli

[0374] This example illustrates preparation of an unglycosylated form ofPRO301, PRO362 or PRO245 by recombinant expression in E. coli.

[0375] The DNA sequence encoding PRO301, PRO362 or PRO245 is initiallyamplified using selected PCR primers. The primers should containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector. A variety of expression vectorsmay be employed. An example of a suitable vector is pBR322 (derived fromE. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the PRO301, PRO362 or PRO245coding region, lambda transcriptional terminator, and an argU gene.

[0376] The ligation mixture is then used to transform a selected E. colistrain using the methods described in Sambrook et al., supra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

[0377] Selected clones can be grown overnight in liquid culture mediumsuch as LB broth supplemented with antibiotics. The overnight culturemay subsequently be used to inoculate a larger scale culture. The cellsare then grown to a desired optical density, during which the expressionpromoter is turned on.

[0378] After culturing the cells for several more hours, the cells canbe harvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO301, PRO362 or PRO245 protein can then bepurified using a metal chelating column under conditions that allowtight binding of the protein.

[0379] PRO301 was expressed in E. coli in a poly-His tagged form, usingthe following procedure. The DNA encoding PRO301 was initially amplifiedusing selected PCR primers. The primers contained restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences were then ligated into anexpression vector, which was used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).Transformants were first grown in LB containing 50 mg/ml carbenicillinat 30° C. with shaking until an O.D.600 of 3-5 was reached. Cultureswere then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate. 2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples were removedto verify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets were frozen untilpurification and refolding.

[0380]E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) wasresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionwas stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution wascentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant was diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. Depending the clarified extract was loaded onto a 5ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelatecolumn buffer. The column was washed with additional buffer containing50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein waseluted with buffer containing 250 mM imidazole. Fractions containing thedesired protein were pooled and stored at 4° C. Protein concentrationwas estimated by its absorbance at 280 nm using the calculatedextinction coefficient based on its amino acid sequence.

[0381] The protein was refolded by diluting sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes were chosen so that the final protein concentration was between50 to 100 micrograms/ml. The refolding solution was stirred gently at 4°C. for 12-36 hours. The refolding reaction was quenched by the additionof TFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution was filtered through a0.22 micron filter and acetonitrile was added to 2-10% finalconcentration. The refolded protein was chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance were analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein were pooled.Generally, the properly refolded species of most proteins are eluted atthe lowest concentrations of acetonitrile since those species are themost compact with their hydrophobic interiors shielded from interactionwith the reversed phase resin. Aggregated species are usually eluted athigher acetonitrile concentrations. In addition to resolving misfoldedforms of proteins from the desired form, the reversed phase step alsoremoves endotoxin from the samples.

[0382] Fractions containing the desired folded PRO301 protein,respectively, were pooled and the acetonitrile removed using a gentlestream of nitrogen directed at the solution. Proteins were formulatedinto 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol bydialysis or by gel filtration using G25 Superfine (Pharmacia) resinsequilibrated in the formulation buffer and sterile filtered.

Example 14 Expression of PRO301, PRO362 or PRO245 in Mammalian Cells

[0383] This example illustrates preparation of a glycosylated form of aPRO301, PRO362 or PRO245 by recombinant expression in mammalian cells.

[0384] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), isemployed as the expression vector. Optionally, the PRO301, PRO362 orPRO245 DNA is ligated into pRK5 with selected restriction enzymes toallow insertion of the PRO301, PRO362 or PRO245 DNA using ligationmethods such as described in Sambrook et al., supra. The resultingvector is called pRK5-PRO301, pRK5-PRO362 or pRK5-PRO245, respectively.

[0385] In one embodiment, the selected host cells may be 293 cells.Human 293 cells (ATCC CCL 1573) are grown to confluence in tissueculture plates in medium such as DMEM supplemented with fetal calf serumand optionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO301, pRK5-PRO362 or pRK5-PRO245 DNA is mixed with about 1 μg DNAencoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] anddissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. Tothis mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mMNaCl, 1.5 mM NaPO₄, and a precipitate is allowed to form for 10 minutesat 25° C. The precipitate is suspended and added to the 293 cells andallowed to settle for about four hours at 37° C. The culture medium isaspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds.The 293 cells are then washed with serum free medium, fresh medium isadded and the cells are incubated for about 5 days.

[0386] Approximately 24 hours after the transfections, the culturemedium is removed and replaced with culture medium (alone) or culturemedium containing 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of PRO301, PRO362 or PRO245 polypeptide. Thecultures containing transfected cells may undergo further incubation (inserum free medium) and the medium is tested in selected bioassays.

[0387] In an alternative technique, PRO301, PRO362 or PRO245 DNA may beintroduced into 293 cells transiently using the dextran sulfate methoddescribed by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981).293 cells are grown to maximal density in a spinner flask and 700 μgpRK5-PRO301, pRK5-PRO362 or pRK5-PRO245 DNA is added. The cells arefirst concentrated from the spinner flask by centrifugation and washedwith PBS. The DNA-dextran precipitate is incubated on the cell pelletfor four hours. The cells are treated with 20% glycerol for 90 seconds,washed with tissue culture medium, and re-introduced into the spinnerflask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1μg/ml bovine transferrin. After about four days, the conditioned mediais centrifuged and filtered to remove cells and debris. The samplecontaining expressed PRO301, PRO362 or PRO245 can then be concentratedand purified by any selected method, such as dialysis and/or columnchromatography.

[0388] In another embodiment, PRO301, PRO362 or PRO245 can be expressedin CHO cells. The pRK5-PRO301, pRK5-PRO362 or pRK5-PRO245 can betransfected into CHO cells using known reagents such as CaPO₄ orDEAE-dextran. As described above, the cell cultures can be incubated,and the medium replaced with culture medium (alone) or medium containinga radiolabel such as ³⁵S-methionine. After determining the presence ofPRO301, PRO362 or PRO245 polypeptide, the culture medium may be replacedwith serum free medium. Preferably, the cultures are incubated for about6 days, and then the conditioned medium is harvested. The mediumcontaining the expressed PRO301, PRO362 or PRO245 can then beconcentrated and purified by any selected method.

[0389] Epitope-tagged PRO301, PRO362 or PRO245 may also be expressed inhost CHO cells. The PRO301, PRO362 or PRO245 may be subcloned out of thepRK5 vector. The subclone insert can undergo PCR to fuse in frame with aselected epitope tag such as a poly-his tag into a Baculovirusexpression vector. The poly-his tagged PRO301, PRO362 or PRO245 insertcan then be subcloned into a SV40 driven vector containing a selectionmarker such as DHFR for selection of stable clones. Finally, the CHOcells can be transfected (as described above) with the SV40 drivenvector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedPRO301, PRO362 or PRO245 can then be concentrated and purified by anyselected method, such as by Ni²⁺-chelate affinity chromatography.

[0390] PRO301, PRO362 and PRO245 were expressed in CHO cells by both atransient and stable expression procedure.

[0391] Stable expression in CHO cells was performed using the followingprocedure. The proteins were expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins were fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or as a poly-His tagged form.

[0392] Following PCR amplification, the respective DNAs were subclonedin a CHO expression vector using standard techniques as described inAusubel et al., Current Protocols of Molecular Biology, Unit 3.16, JohnWiley and Sons (1997). CHO expression vectors are constructed to havecompatible restriction sites 5

and 3

of the DNA of interest to allow the convenient shuttling of cDNA

s. The vector used expression in CHO cells is as described in Lucas etal., Nucl. Acids Res. 24: 9 (1774-1779 (1996), and uses the SV40 earlypromoter/enhancer to drive expression of the cDNA of interest anddihydrofolate reductase (DHFR). DHFR expression permits selection forstable maintenance of the plasmid following transfection.

[0393] Twelve micrograms of the desired plasmid DNA were introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect

(Quiagen), Dosper

or Fugene

(Boehringer Mannheim). The cells were grown and described in Lucas etal., supra. Approximately 3×10⁻⁷ cells are frozen in an ampule forfurther growth and production as described below.

[0394] The ampules containing the plasmid DNA were thawed by placementinto water bath and mixed by vortexing. The contents were pipetted intoa centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant was aspirated and the cells wereresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells were then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells were transferred into a 250 mL spinner filled with 150mL selective growth medium and incubated at 37° C. After another 2-3days, a 250 mL 500 mL and 2000 mL spinners were seeded with 3×10⁵cells/mL. The cell media was exchanged with fresh media bycentrifugation and resuspension in production medium. Although anysuitable CHO media may be employed, a production medium described inU.S. Pat. No. 5,122,469, issued Jun. 16, 1992 was actually used. 3Lproduction spinner is seeded at 1.2×10⁶ cells/mL. On day 0, the cellnumber and pH were determined. On day 1, the spinner was sampled andsparging with filtered air was commenced. On day 2, the spinner wassampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucoseand 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, DowCorning 365 Medical Grade Emulsion). Throughout the production, pH wasadjusted as necessary to keep at around 7.2. After 10 days, or untilviability dropped below 70%, the cell culture was harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

[0395] For the poly-His tagged constructs, the proteins were purifiedusing a Ni-NTA column (Qiagen). Before purification, imidazole was addedto the conditioned media to a concentration of 5 mM. The conditionedmedia was pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes,pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rateof 4-5 ml/min. at 4° C. After loading, the column was washed withadditional equilibration buffer and the protein eluted withequilibration buffer containing 0.25 M imidazole. The highly purifiedprotein was subsequently desalted into a storage buffer containing 10 mMHepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine(Pharmacia) column and stored at −80° C.

[0396] Immunoadhesin (Fc containing) constructs of were purified fromthe conditioned media as follows. The conditioned medium was pumped ontoa 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mMNa phosphate buffer, pH 6.8. After loading, the column was washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein was immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein was subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity was assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

[0397] PRO301, PRO362 and PRO245 was also produced by transientexpression in COS cells.

Example 15 Expression of PRO301, PRO362 or PRO245 in Yeast

[0398] The following method describes recombinant expression of PRO301,PRO362 or PRO245 in yeast.

[0399] First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO301, PRO362 or PRO245 from the ADH2/GAPDHpromoter. DNA encoding PRO301, PRO362 or PRO245, a selected signalpeptide and the promoter is inserted into suitable restriction enzymesites in the selected plasmid to direct intracellular expression ofPRO301, PRO362 or PRO245. For secretion, DNA encoding PRO301, PRO362 orPRO245 can be cloned into the selected plasmid, together with DNAencoding the ADH2/GAPDH promoter, the yeast alpha-factor secretorysignal/leader sequence, and linker sequences (if needed) for expressionof PRO301, PRO362 or PRO245.

[0400] Yeast cells, such as yeast strain AB110, can then be transformedwith the expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0401] Recombinant PRO301, PRO362 or PRO245 can subsequently be isolatedand purified by removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing PRO301, PRO362 or PRO245may further be purified using selected column chromatography resins.

Example 16 Expression of PRO301, PRO362 or PRO245 inBaculovirus-Infected Insect Cells

[0402] The following method describes recombinant expression of PRO301,PRO362 or PRO245 in Baculovirus-infected insect cells.

[0403] The PRO301, PRO362 or PRO245 is fused upstream of an epitope tagcontained with a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thePRO301, PRO362 or PRO245 or the desired portion of the PRO301, PRO362 orPRO245 (such as the sequence encoding the extracellular domain of atransmembrane protein) is amplified by PCR with primers complementary tothe 5′ and 3′ regions. The 5′ primer may incorporate flanking (selected)restriction enzyme sites. The product is then digested with thoseselected restriction enzymes and subcloned into the expression vector.

[0404] Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

[0405] Expressed poly-his tagged PRO301, PRO362 or PRO245 can then bepurified, for example, by Ni²⁺-chelate affinity chromatography asfollows. Extracts are prepared from recombinant virus-infected Sf9 cellsas described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9;12.5 mM MgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP40; 0.4 M KCl), andsonicated twice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 Fm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO301, PRO362 or PRO245 are pooledand dialyzed against loading buffer.

[0406] Alternatively, purification of the IgG tagged (or Fc tagged)PRO301, PRO362 or PRO245 can be performed using known chromatographytechniques, including for instance, Protein A or protein G columnchromatography.

[0407] PRO301, PRO362 and PRO245 were expressed in baculovirus infectedSf9 insect cells. While the expression was actually performed in a 0.5-2L scale, it can be readily scaled up for larger (e.g. 8 L) preparations.The proteins were expressed as an IgG construct (immunoadhesin), inwhich the protein extracellular region was fused to an IgG1 constantregion sequence containing the hinge, CH2 and CH3 domains and/or inpoly-His tagged forms.

[0408] Following PCR amplification, the respective coding sequences weresubcloned into a baculovirus expression vector (pb.PH.IgG for IgGfusions and pb.PH.His.c for poly-His tagged proteins), and the vectorand Baculogold

baculovirus DNA (Pharmingen) were co-transfected into 105 Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711), using Lipofectin (Gibco BRL).pb.PH.IgG and pb.PH.His are modifications of the commercially availablebaculovirus expression vector pVL1393 (Pharmingen), with modifiedpolylinker regions to include the His or Fc tag sequences. The cellswere grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone).Cells were incubated for 5 days at 28° C. The supernatant was harvestedand subsequently used for the first viral amplification by infecting Sf9cells in Hink's TNM-FH medium supplemented with 10% FBS at anapproximate multiplicity of infection (MOI) of 10. Cells were incubatedfor 3 days at 28° C. The supernatant was harvested and the expression ofthe constructs in the baculovirus expression vector was determined bybatch binding of 1 ml of supernatant to 25 mL of Ni-NTA beads (QIAGEN)for histidine tagged proteins or Protein-A Sepharose CL4B beads(Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysiscomparing to a known concentration of protein standard by Coomassie bluestaining.

[0409] The first viral amplification supernatant was used to infect aspinner culture (500 ml) of Sf9 cells grown in ESF-921 medium(Expression Systems LLC) at an approximate MOI of 0.1. Cells wereincubated for 3 days at 28° C. The supernatant was harvested andfiltered. Batch binding and SDS-PAGE analysis was repeated, asnecessary, until expression of the spinner culture was confirmed.

[0410] The conditioned medium from the transfected cells (0.5 to 3 L)was harvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteinconstruct were purified using a Ni-NTA column (Qiagen). Beforepurification, imidazole was added to the conditioned media to aconcentration of 5 mM. The conditioned media were pumped onto a 6 mlNi-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4° C. Afterloading, the column was washed with additional equilibration buffer andthe protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein was subsequently desalted into astorage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at −80° C.

[0411] Immunoadhesin (Fc containing) constructs of proteins werepurified from the conditioned media as follows. The conditioned mediawere pumped onto a 5 ml Protein A column (Pharmacia) which had beenequilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, thecolumn was washed extensively with equilibration buffer before elutionwith 100 mM citric acid, pH 3.5. The eluted protein was immediatelyneutralized by collecting 1 ml fractions into tubes containing 275 mL of1 M Tris buffer, pH 9. The highly purified protein was subsequentlydesalted into storage buffer as described above for the poly-His taggedproteins. The homogeneity of the proteins was verified by SDSpolyacrylamide gel (PEG) electrophoresis and N-terminal amino acidsequencing by Edman degradation.

[0412] PRO301, PRO362 and PRO245 were also expressed in baculovirusinfected High-5 cells using an analogous procedure. High-5 cells weregrown to a confluency of 50% at 27° C., no CO₂, no penicillin and nostreptomycin. For each 150 mm plate, 30 μg of pIE based vectorcontaining PRO301, PRO362 or PRO245 was mixed with 1 ml Ex-Cell medium(Media: Ex-cell 401, 1/100 L-Glu JRH Biosciences, #14401-78P, note:medium is light sensitive), and in a separate tube, 100 μl of CellFectin (GibcoBRL #10362-010) was mixed with 1 ml of Ec-Cell medium. ThepIE1-1 and pIE1-2 vectors are designed for constitutive expression ofrecombinant proteins from the baculovirus ie 1 promoter instably-transformed insect cells (Cartier, J. L., et al., J. Virol. 68,7728-7737)(1994). The plasmids differ only in the orientation of themultiple cloning sites and contain all promoter sequences known to beimportant for ie 1-mediated gene expression in uninfected insect cellsas well as the hr5 enhancer element. pIE1-1 and pIE1-2 include the ieltranslation initiation site and can be used to produce fusion proteins.

[0413] The two solutions were combined and allowed to incubate at roomtemperature for 15 minutes. 8 ml of Ex-Cell media was added to the 2 mlof DNA/CellFectin mix and is layered on High-5 cells previously washedwith Ex-Cell media. The plate was incubated in darkness for 1 hour atroom temperature. The DNA/CellFectin mix was aspirated, and the cellswashed once with Ex-Cell to remove excess Cellfectin. Fresh Ex-cellmedium (30 ml) was added and the cells incubated for 3 days at 28° C.The supernatent was harvested and the expression of PRO301, PRO362 orPRO245 was determined by batch binding in a manner simliar to thatdescribed for Sf9 cells.

Example 17 Preparation of Antibodies that Bind PRO301, PRO362 and PRO245

[0414] This example illustrates preparation of monoclonal antibodieswhich can specifically bind PRO301, PRO362 and PRO245.

[0415] Techniques for producing the monoclonal antibodies are known inthe art and are described, for instance, in Goding, supra. Immunogensthat may be employed include purified PRO301, PRO362 and PRO245, fusionproteins containing PRO301, PRO362 and PRO245, and cells expressingrecombinant PRO301, PRO362 and PRO245 on the cell surface. Selection ofthe immunogen can be made by the skilled artisan without undueexperimentation.

[0416] Mice, such as Balb/c, are immunized with the PRO301, PRO362 andPRO245 immunogen emulsified in complete Freund's adjuvant and injectedsubcutaneously or intraperitoneally in an amount from 1-100 micrograms.Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (RibiImmunochemical Research, Hamilton, Mont.) and injected into the animal'shind foot pads. The immunized mice are then boosted 10 to 12 days laterwith additional immunogen emulsified in the selected adjuvant.Thereafter, for several weeks, the mice may also be boosted withadditional immunization injections. Serum samples may be periodicallyobtained from the mice by retro-orbital bleeding for testing in ELISAassays to detect PRO301, PRO362 and PRO245 antibodies.

[0417] After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO301, PRO362 and PRO245. Three to four days later, themice are sacrificed and the spleen cells are harvested. The spleen cellsare then fused (using 35% polyethylene glycol) to a selected murinemyeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL 1597.The fusions generate hybridoma cells which can then be plated in 96 welltissue culture plates containing HAT (hypoxanthine, aminopterin, andthymidine) medium to inhibit proliferation of non-fused cells, myelomahybrids, and spleen cell hybrids.

[0418] The hybridoma cells will be screened in an ELISA for reactivityagainst PRO301, PRO362 and PRO245. Determination of “positive” hybridomacells secreting the desired monoclonal antibodies against PRO301, PRO362and PRO245 is within the skill in the art.

[0419] The positive hybridoma cells can be injected intraperitoneallyinto syngeneic Balb/c mice to produce ascites containing theanti-PRO301, anti-PRO362 or anti-PRO245 monoclonal antibodies.Alternatively, the hybridoma cells can be grown in tissue culture flasksor roller bottles. Purification of the monoclonal antibodies produced inthe ascites can be accomplished using ammonium sulfate precipitation,followed by gel exclusion chromatography. Alternatively, affinitychromatography based upon binding of antibody to protein A or protein Gcan be employed.

[0420] Deposit of Material

[0421] The following materials have been deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC): Designation ATCC Dep. No. Deposit Date pRK5-based plasmid 209432Nov. 7, 1997 DNA40628-1216 DNA45416-1251 209620 Feb. 5, 1998DNA35638-1141 209265 Sep. 16, 1997

[0422] These deposits were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of a viable culture of thedeposit for 30 years from the date of deposit. The deposit will be madeavailable by ATCC under the terms of the Budapest Treaty, and subject toan agreement between Genentech, Inc. and ATCC, which assures permanentand unrestricted availability of the progeny of the culture of thedeposit to the public upon issuance of the pertinent U.S. patent or uponlaying open to the public of any U.S. or foreign patent application,whichever comes first, and assures availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 USC '122 and the Conmissioner's rulespursuant thereto (including 37 CFR § 1.14 with particular reference to886 OG 638).

[0423] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

[0424] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1 30 1 299 PRT Homo sapiens 1 Met Gly Thr Lys Ala Gln Val Glu Arg LysLeu Leu Cys Leu Phe 1 5 10 15 Ile Leu Ala Ile Leu Leu Cys Ser Leu AlaLeu Gly Ser Val Thr 20 25 30 Val His Ser Ser Glu Pro Glu Val Arg Ile ProGlu Asn Asn Pro 35 40 45 Val Lys Leu Ser Cys Ala Tyr Ser Gly Phe Ser SerPro Arg Val 50 55 60 Glu Trp Lys Phe Asp Gln Gly Asp Thr Thr Arg Leu ValCys Tyr 65 70 75 Asn Asn Lys Ile Thr Ala Ser Tyr Glu Asp Arg Val Thr PheLeu 80 85 90 Pro Thr Gly Ile Thr Phe Lys Ser Val Thr Arg Glu Asp Thr Gly95 100 105 Thr Tyr Thr Cys Met Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly110 115 120 Glu Val Lys Val Lys Leu Ile Val Leu Val Pro Pro Ser Lys Pro125 130 135 Thr Val Asn Ile Pro Ser Ser Ala Thr Ile Gly Asn Arg Ala Val140 145 150 Leu Thr Cys Ser Glu Gln Asp Gly Ser Pro Pro Ser Glu Tyr Thr155 160 165 Trp Phe Lys Asp Gly Ile Val Met Pro Thr Asn Pro Lys Ser Thr170 175 180 Arg Ala Phe Ser Asn Ser Ser Tyr Val Leu Asn Pro Thr Thr Gly185 190 195 Glu Leu Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr200 205 210 Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser Asn215 220 225 Ala Val Arg Met Glu Ala Val Glu Arg Asn Val Gly Val Ile Val230 235 240 Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Ile Leu Val Phe245 250 255 Gly Ile Trp Phe Ala Tyr Ser Arg Gly His Phe Asp Arg Thr Lys260 265 270 Lys Gly Thr Ser Ser Lys Lys Val Ile Tyr Ser Gln Pro Ser Ala275 280 285 Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu Val 290295 2 321 PRT Homo sapiens 2 Met Gly Ile Leu Leu Gly Leu Leu Leu Leu GlyHis Leu Thr Val 1 5 10 15 Asp Thr Tyr Gly Arg Pro Ile Leu Glu Val ProGlu Ser Val Thr 20 25 30 Gly Pro Trp Lys Gly Asp Val Asn Leu Pro Cys ThrTyr Asp Pro 35 40 45 Leu Gln Gly Tyr Thr Gln Val Leu Val Lys Trp Leu ValGln Arg 50 55 60 Gly Ser Asp Pro Val Thr Ile Phe Leu Arg Asp Ser Ser GlyAsp 65 70 75 His Ile Gln Gln Ala Lys Tyr Gln Gly Arg Leu His Val Ser His80 85 90 Lys Val Pro Gly Asp Val Ser Leu Gln Leu Ser Thr Leu Glu Met 95100 105 Asp Asp Arg Ser His Tyr Thr Cys Glu Val Thr Trp Gln Thr Pro 110115 120 Asp Gly Asn Gln Val Val Arg Asp Lys Ile Thr Glu Leu Arg Val 125130 135 Gln Lys Leu Ser Val Ser Lys Pro Thr Val Thr Thr Gly Ser Gly 140145 150 Tyr Gly Phe Thr Val Pro Gln Gly Met Arg Ile Ser Leu Gln Cys 155160 165 Gln Ala Arg Gly Ser Pro Pro Ile Ser Tyr Ile Trp Tyr Lys Gln 170175 180 Gln Thr Asn Asn Gln Glu Pro Ile Lys Val Ala Thr Leu Ser Thr 185190 195 Leu Leu Phe Lys Pro Ala Val Ile Ala Asp Ser Gly Ser Tyr Phe 200205 210 Cys Thr Ala Lys Gly Gln Val Gly Ser Glu Gln His Ser Asp Ile 215220 225 Val Lys Phe Val Val Lys Asp Ser Ser Lys Leu Leu Lys Thr Lys 230235 240 Thr Glu Ala Pro Thr Thr Met Thr Tyr Pro Leu Lys Ala Thr Ser 245250 255 Thr Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr 260265 270 Leu Gly Glu Thr Ser Ala Gly Pro Gly Lys Ser Leu Pro Val Phe 275280 285 Ala Ile Ile Leu Ile Ile Ser Leu Cys Cys Met Val Val Phe Thr 290295 300 Met Ala Tyr Ile Met Leu Cys Arg Lys Thr Ser Gln Gln Glu His 305310 315 Val Tyr Glu Ala Ala Arg 320 3 390 DNA Homo Sapien 3 cttcttgccaactggtatca ccttcaagtc cgtgacacgg gaagacactg 50 ggacatacac ttgtatggtctctgaggaag gcggcaacag ctatggggag 100 gtcaaggtca agctcatcgt gcttgtgcctccatccaagc ctacagttaa 150 catcccctcc tctgccacca ttgggaaccg ggcagtgctgacatgctcag 200 aacaagatgg ttccccacct tctgaataca cctggttcaa agatgggata250 gtgatgccta cgaatcccaa aagcacccgt gccttcagca actcttccta 300tgtcctgaat cccacaacag gagagctggt ctttgatccc ctgtcagcct 350 ctgatactggagaatacagc tgtgaggcac ggaatgggta 390 4 726 DNA Homo Sapien 4 tctcagtcccctcgctgtag tcgcggagct gtgttctgtt tcccaggagt 50 ccttcggcgg ctgttgtgctcaggtgcgcc tgatcgcgat ggggacaaag 100 gcgcaagctc gagaggaaac tgttgtgcctcttcatattg gcgatcctgt 150 tgtgctccct ggcattgggc agtgttacag ttgcactcttctgaacctga 200 agtcagaatt cctgagaata atcctgtgaa gttgtcctgt gcctactcgg250 gcttttcttc tccccgtgtg gagtggaagt ttgaccaagg agacaccacc 300agactcgttt gctataataa caagatcaca gcttcctatg aggaccgggt 350 gaccttcttgccaactggta tcaccttcaa gtccgtgaca cgggaagaca 400 ctgggacata cacttgtatggtctctgagg aaggcggcaa cagctatggg 450 gaggtcaagg tcaagctcat cgtgcttgtgcctccatcca agcctacagt 500 taacatcccc tcctctgcca ccattgggaa ccgggcagtgctgacatgct 550 cagaacaaga tggttcccca ccttctgaat acacctggtt caaagatggg600 atagtgatgc ctacgaatcc caaaagcacc cgtgccttca gcaactcttc 650ctatgtcctg aatcccacaa caggagagct ggtctttgat cccctgtcag 700 cctctgatactggagaatac agctgt 726 5 1503 DNA Homo Sapien 5 gcaggcaaag taccagggccgcctgcatgt gagccacaag gttccaggag 50 atgtatccct ccaattgagc accctggagatggatgaccg gagccactac 100 acgtgtgaag tcacctggca gactcctgat ggcaaccaagtcgtgagaga 150 taagattact gagctccgtg tccagaaact ctctgtctcc aagcccacag200 tgacaactgg cagcggttat ggcttcacgg tgccccaggg aatgaggatt 250agccttcaat gccagggttc ggggttctcc tcccatcagt tatatttggt 300 ataagcaacagactaataac cagggaaccc atcaaagtag caaccctaag 350 taccttactc ttcaagcctgcggtgatagc cgactcaggc tcctatttct 400 gcactgccaa gggccaggtt ggctctgagcagcacagcga cattgtgaag 450 tttgtggtca aagactcctc aaagctactc aagaccaagactgaggcacc 500 tacaaccatg acatacccct tgaaagcaac atctacagtg aagcagtcct550 gggactggac cactgacatg gatggctacc ttggagagac cagtgctggg 600ccaggaaaga gcctgcctgt ctttgccatc atcctcatca tctccttgtg 650 ctgtatggtggtttttacca tggcctatat catgctctgt cggaagacat 700 cccaacaaga gcatgtctacgaagcagcca gggcacatgc cagagaggcc 750 aacgactctg gagaaaccat gagggtggccatcttcgcaa gtggctgctc 800 cagtgatgag ccaacttccc agaatctggg gcaacaactactctgatgag 850 ccctgcatag gacaggagta ccagatcatc gcccagatca atggcaacta900 cgcccgcctg ctggacacag ttcctctgga ttatgagttt ctggccactg 950agggcaaaag tgtctgttaa aaatgcccca ttaggccagg atctgctgac 1000 ataattgcctagtcagtcct tgccttctgc atggccttct tccctgctac 1050 ctctcttcct ggatagcccaaagtgtccgc ctaccaacac tggagccgct 1100 gggagtcact ggctttgccc tggaatttgccagatgcatc tcaagtaagc 1150 cagctgctgg atttggctct gggcccttct agtatctctgccgggggctt 1200 ctggtactcc tctctaaata ccagagggaa gatgcccata gcactaggac1250 ttggtcatca tgcctacaga cactattcaa ctttggcatc ttgccaccag 1300aagacccgag gggaggctca gctctgccag ctcagaggac cagctatatc 1350 caggatcatttctctttctt cagggccaga cagcttttaa ttgaaattgt 1400 tatttcacag gccagggttcagttctgctc ctccactata agtctaatgt 1450 tctgactctc tcctggtgct caataaatatctaatcataa cagcaaaaaa 1500 aaa 1503 6 319 PRT Homo sapiens 6 Met Val GlyLys Met Trp Pro Val Leu Trp Thr Leu Cys Ala Val 1 5 10 15 Arg Val ThrVal Asp Ala Ile Ser Val Glu Thr Pro Gln Asp Val 20 25 30 Leu Arg Ala SerGln Gly Lys Ser Val Thr Leu Pro Cys Thr Tyr 35 40 45 His Thr Ser Thr SerSer Arg Glu Gly Leu Ile Gln Trp Asp Lys 50 55 60 Leu Leu Leu Thr His ThrGlu Arg Val Val Ile Trp Pro Phe Ser 65 70 75 Asn Lys Asn Tyr Ile His GlyGlu Leu Tyr Lys Asn Arg Val Ser 80 85 90 Ile Ser Asn Asn Ala Glu Gln SerAsp Ala Ser Ile Thr Ile Asp 95 100 105 Gln Leu Thr Met Ala Asp Asn GlyThr Tyr Glu Cys Ser Val Ser 110 115 120 Leu Met Ser Asp Leu Glu Gly AsnThr Lys Ser Arg Val Arg Leu 125 130 135 Leu Val Leu Val Pro Pro Ser LysPro Glu Cys Gly Ile Glu Gly 140 145 150 Glu Thr Ile Ile Gly Asn Asn IleGln Leu Thr Cys Gln Ser Lys 155 160 165 Glu Gly Ser Pro Thr Pro Gln TyrSer Trp Lys Arg Tyr Asn Ile 170 175 180 Leu Asn Gln Glu Gln Pro Leu AlaGln Pro Ala Ser Gly Gln Pro 185 190 195 Val Ser Leu Lys Asn Ile Ser ThrAsp Thr Ser Gly Tyr Tyr Ile 200 205 210 Cys Thr Ser Ser Asn Glu Glu GlyThr Gln Phe Cys Asn Ile Thr 215 220 225 Val Ala Val Arg Ser Pro Ser MetAsn Val Ala Leu Tyr Val Gly 230 235 240 Ile Ala Val Gly Val Val Ala AlaLeu Ile Ile Ile Gly Ile Ile 245 250 255 Ile Tyr Cys Cys Cys Cys Arg GlyLys Asp Asp Asn Thr Glu Asp 260 265 270 Lys Glu Asp Ala Arg Pro Asn ArgGlu Ala Tyr Glu Glu Pro Pro 275 280 285 Glu Gln Leu Arg Glu Leu Ser ArgGlu Arg Glu Glu Glu Asp Asp 290 295 300 Tyr Arg Gln Glu Glu Gln Arg SerThr Gly Arg Glu Ser Pro Asp 305 310 315 His Leu Asp Gln 7 2181 DNA Homosapiens 7 cccacgcgtc cgcccacgcg tccgcccacg ggtccgccca cgcgtccggg 50ccaccagaag tttgagcctc tttggtagca ggaggctgga agaaaggaca 100 gaagtagctctggctgtgat ggggatctta ctgggcctgc tactcctggg 150 gcacctaaca gtggacacttatggccgtcc catcctggaa gtgccagaga 200 gtgtaacagg accttggaaa ggggatgtgaatcttccctg cacctatgac 250 cccctgcaag gctacaccca agtcttggtg aagtggctggtacaacgtgg 300 ctcagaccct gtcaccatct ttctacgtga ctcttctgga gaccatatcc350 agcaggcaaa gtaccagggc cgcctgcatg tgagccacaa ggttccagga 400gatgtatccc tccaattgag caccctggag atggatgacc ggagccacta 450 cacgtgtgaagtcacctggc agactcctga tggcaaccaa gtcgtgagag 500 ataagattac tgagctccgtgtccagaaac tctctgtctc caagcccaca 550 gtgacaactg gcagcggtta tggcttcacggtgccccagg gaatgaggat 600 tagccttcaa tgccaggctc ggggttctcc tcccatcagttatatttggt 650 ataagcaaca gactaataac caggaaccca tcaaagtagc aaccctaagt700 accttactct tcaagcctgc ggtgatagcc gactcaggct cctatttctg 750cactgccaag ggccaggttg gctctgagca gcacagcgac attgtgaagt 800 ttgtggtcaaagactcctca aagctactca agaccaagac tgaggcacct 850 acaaccatga cataccccttgaaagcaaca tctacagtga agcagtcctg 900 ggactggacc actgacatgg atggctaccttggagagacc agtgctgggc 950 caggaaagag cctgcctgtc tttgccatca tcctcatcatctccttgtgc 1000 tgtatggtgg tttttaccat ggcctatatc atgctctgtc ggaagacatc1050 ccaacaagag catgtctacg aagcagccag gtaagaaagt ctctcctctt 1100ccatttttga ccccgtccct gccctcaatt ttgattactg gcaggaaatg 1150 tggaggaaggggggtgtggc acagacccaa tcctaaggcc ggaggccttc 1200 agggtcagga catagctgccttccctctct caggcacctt ctgaggttgt 1250 tttggccctc tgaacacaaa ggataatttagatccatctg ccttctgctt 1300 ccagaatccc tgggtggtag gatcctgata attaattggcaagaattgag 1350 gcagaagggt gggaaaccag gaccacagcc ccaagtccct tcttatgggt1400 ggtgggctct tgggccatag ggcacatgcc agagaggcca acgactctgg 1450agaaaccatg agggtggcca tcttcgcaag tggctgctcc agtgatgagc 1500 caacttcccagaatctgggc aacaactact ctgatgagcc ctgcatagga 1550 caggagtacc agatcatcgcccagatcaat ggcaactacg cccgcctgct 1600 ggacacagtt cctctggatt atgagtttctggccactgag ggcaaaagtg 1650 tctgttaaaa atgccccatt aggccaggat ctgctgacataattgcctag 1700 tcagtccttg ccttctgcat ggccttcttc cctgctacct ctcttcctgg1750 atagcccaaa gtgtccgcct accaacactg gagccgctgg gagtcactgg 1800ctttgccctg gaatttgcca gatgcatctc aagtaagcca gctgctggat 1850 ttggctctgggcccttctag tatctctgcc gggggcttct ggtactcctc 1900 tctaaatacc agagggaagatgcccatagc actaggactt ggtcatcatg 1950 cctacagaca ctattcaact ttggcatcttgccaccagaa gacccgaggg 2000 aggctcagct ctgccagctc agaggaccag ctatatccaggatcatttct 2050 ctttcttcag ggccagacag cttttaattg aaattgttat ttcacaggcc2100 agggttcagt tctgctcctc cactataagt ctaatgttct gactctctcc 2150tggtgctcaa taaatatcta atcataacag c 2181 8 1295 DNA Homo sapiens 8cccagaagtt caagggcccc cggcctcctg cgctcctgcc gccgggaccc 50 tcgacctcctcagagcagcc ggctgccgcc ccgggaagat ggcgaggagg 100 agccgccacc gcctcctcctgctgctgctg cgctacctgg tggtcgccct 150 gggctatcat aaggcctatg ggttttctgccccaaaagac caacaagtag 200 tcacagcagt agagtaccaa gaggctattt tagcctgcaaaaccccaaag 250 aagactgttt cctccagatt agagtggaag aaactgggtc ggagtgtctc300 ctttgtctac tatcaacaga ctcttcaagg tgattttaaa aatcgagctg 350agatgataga tttcaatatc cggatcaaaa atgtgacaag aagtgatgcg 400 gggaaatatcgttgtgaagt tagtgcccca tctgagcaag gccaaaacct 450 ggaagaggat acagtcactctggaagtatt agtggctcca gcagttccat 500 catgtgaagt accctcttct gctctgagtggaactgtggt agagctacga 550 tgtcaagaca aagaagggaa tccagctcct gaatacacatggtttaagga 600 tggcatccgt ttgctagaaa atcccagact tggctcccaa agcaccaaca650 gctcatacac aatgaataca aaaactggaa ctctgcaatt taatactgtt 700tccaaactgg acactggaga atattcctgt gaagcccgca attctgttgg 750 atatcgcaggtgtcctggga aacgaatgca agtagatgat ctcaacataa 800 gtggcatcat agcagccgtagtagttgtgg ccttagtgat ttccgtttgt 850 ggccttggtg tatgctatgc tcagaggaaaggctactttt caaaagaaac 900 ctccttccag aagagtaatt cttcatctaa agccacgacaatgagtgaaa 950 atgtgcagtg gctcacgcct gtaatcccag cactttggaa ggccgcggcg1000 ggcggatcac gaggtcagga gttctagacc agtctggcca atatggtgaa 1050accccatctc tactaaaata caaaaattag ctgggcatgg tggcatgtgc 1100 ctgcagttccagctgcttgg gagacaggag aatcacttga acccgggagg 1150 cggaggttgc agtgagctgagatcacgcca ctgcagtcca gcctgggtaa 1200 cagagcaaga ttccatctca aaaaataaaataaataaata aataaatact 1250 ggtttttacc tgtagaattc ttacaataaa tatagcttgatattc 1295 9 312 PRT Homo sapiens 9 Met Ala Arg Arg Ser Arg His Arg LeuLeu Leu Leu Leu Leu Arg 1 5 10 15 Tyr Leu Val Val Ala Leu Gly Tyr HisLys Ala Tyr Gly Phe Ser 20 25 30 Ala Pro Lys Asp Gln Gln Val Val Thr AlaVal Glu Tyr Gln Glu 35 40 45 Ala Ile Leu Ala Cys Lys Thr Pro Lys Lys ThrVal Ser Ser Arg 50 55 60 Leu Glu Trp Lys Lys Leu Gly Arg Ser Val Ser PheVal Tyr Tyr 65 70 75 Gln Gln Thr Leu Gln Gly Asp Phe Lys Asn Arg Ala GluMet Ile 80 85 90 Asp Phe Asn Ile Arg Ile Lys Asn Val Thr Arg Ser Asp AlaGly 95 100 105 Lys Tyr Arg Cys Glu Val Ser Ala Pro Ser Glu Gln Gly GlnAsn 110 115 120 Leu Glu Glu Asp Thr Val Thr Leu Glu Val Leu Val Ala ProAla 125 130 135 Val Pro Ser Cys Glu Val Pro Ser Ser Ala Leu Ser Gly ThrVal 140 145 150 Val Glu Leu Arg Cys Gln Asp Lys Glu Gly Asn Pro Ala ProGlu 155 160 165 Tyr Thr Trp Phe Lys Asp Gly Ile Arg Leu Leu Glu Asn ProArg 170 175 180 Leu Gly Ser Gln Ser Thr Asn Ser Ser Tyr Thr Met Asn ThrLys 185 190 195 Thr Gly Thr Leu Gln Phe Asn Thr Val Ser Lys Leu Asp ThrGly 200 205 210 Glu Tyr Ser Cys Glu Ala Arg Asn Ser Val Gly Tyr Arg ArgCys 215 220 225 Pro Gly Lys Arg Met Gln Val Asp Asp Leu Asn Ile Ser GlyIle 230 235 240 Ile Ala Ala Val Val Val Val Ala Leu Val Ile Ser Val CysGly 245 250 255 Leu Gly Val Cys Tyr Ala Gln Arg Lys Gly Tyr Phe Ser LysGlu 260 265 270 Thr Ser Phe Gln Lys Ser Asn Ser Ser Ser Lys Ala Thr ThrMet 275 280 285 Ser Glu Asn Val Gln Trp Leu Thr Pro Val Ile Pro Ala LeuTrp 290 295 300 Lys Ala Ala Ala Gly Gly Ser Arg Gly Gln Glu Phe 305 31010 300 PRT Mus musculus 10 Met Gly Thr Glu Gly Lys Ala Gly Arg Lys LeuLeu Phe Leu Phe 1 5 10 15 Thr Ser Met Ile Leu Gly Ser Leu Val Gln GlyLys Gly Ser Val 20 25 30 Tyr Thr Ala Gln Ser Asp Val Gln Val Pro Glu AsnGlu Ser Ile 35 40 45 Lys Leu Thr Cys Thr Tyr Ser Gly Phe Ser Ser Pro ArgVal Glu 50 55 60 Trp Lys Phe Val Gln Gly Ser Thr Thr Ala Leu Val Cys TyrAsn 65 70 75 Ser Gln Ile Thr Ala Pro Tyr Ala Asp Arg Val Thr Phe Ser Ser80 85 90 Ser Gly Ile Thr Phe Ser Ser Val Thr Arg Lys Asp Asn Gly Glu 95100 105 Tyr Thr Cys Met Val Ser Glu Glu Gly Gly Gln Asn Tyr Gly Glu 110115 120 Val Ser Ile His Leu Thr Val Leu Val Pro Pro Ser Lys Pro Thr 125130 135 Ile Ser Val Pro Ser Ser Val Thr Ile Gly Asn Arg Ala Val Leu 140145 150 Thr Cys Ser Glu His Asp Gly Ser Pro Pro Ser Glu Tyr Ser Trp 155160 165 Phe Lys Asp Gly Ile Ser Met Leu Thr Ala Asp Ala Lys Lys Thr 170175 180 Arg Ala Phe Met Asn Ser Ser Phe Thr Ile Asp Pro Lys Ser Gly 185190 195 Asp Leu Ile Phe Asp Pro Val Thr Ala Phe Asp Ser Gly Glu Tyr 200205 210 Tyr Cys Gln Ala Gln Asn Gly Tyr Gly Thr Ala Met Arg Ser Glu 215220 225 Ala Ala His Met Asp Ala Val Glu Leu Asn Val Gly Gly Ile Val 230235 240 Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Leu Leu Ile Phe 245250 255 Gly Val Trp Phe Ala Tyr Ser Arg Gly Tyr Phe Glu Thr Thr Lys 260265 270 Lys Gly Thr Ala Pro Gly Lys Lys Val Ile Tyr Ser Gln Pro Ser 275280 285 Thr Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu Val 290295 300 11 2181 DNA Homo sapiens 11 cccacgcgtc cgcccacgcg tccgcccacgggtccgccca cgcgtccggg 50 ccaccagaag tttgagcctc tttggtagca ggaggctggaagaaaggaca 100 gaagtagctc tggctgtgat ggggatctta ctgggcctgc tactcctggg150 gcacctaaca gtggacactt atggccgtcc catcctggaa gtgccagaga 200gtgtaacagg accttggaaa ggggatgtga atcttccctg cacctatgac 250 cccctgcaaggctacaccca agtcttggtg aagtggctgg tacaacgtgg 300 ctcagaccct gtcaccatctttctacgtga ctcttctgga gaccatatcc 350 agcaggcaaa gtaccagggc cgcctgcatgtgagccacaa ggttccagga 400 gatgtatccc tccaattgag caccctggag atggatgaccggagccacta 450 cacgtgtgaa gtcacctggc agactcctga tggcaaccaa gtcgtgagag500 ataagattac tgagctccgt gtccagaaac tctctgtctc caagcccaca 550gtgacaactg gcagcggtta tggcttcacg gtgccccagg gaatgaggat 600 tagccttcaatgccaggctc ggggttctcc tcccatcagt tatatttggt 650 ataagcaaca gactaataaccaggaaccca tcaaagtagc aaccctaagt 700 accttactct tcaagcctgc ggtgatagccgactcaggct cctatttctg 750 cactgccaag ggccaggttg gctctgagca gcacagcgacattgtgaagt 800 ttgtggtcaa agactcctca aagctactca agaccaagac tgaggcacct850 acaaccatga catacccctt gaaagcaaca tctacagtga agcagtcctg 900ggactggacc actgacatgg atggctacct tggagagacc agtgctgggc 950 caggaaagagcctgcctgtc tttgccatca tcctcatcat ctccttgtgc 1000 tgtatggtgg tttttaccatggcctatatc atgctctgtc ggaagacatc 1050 ccaacaagag catgtctacg aagcagccaggtaagaaagt ctctcctctt 1100 ccatttttga ccccgtccct gccctcaatt ttgattactggcaggaaatg 1150 tggaggaagg ggggtgtggc acagacccaa tcctaaggcc ggaggccttc1200 agggtcagga catagctgcc ttccctctct caggcacctt ctgaggttgt 1250tttggccctc tgaacacaaa ggataattta gatccatctg ccttctgctt 1300 ccagaatccctgggtggtag gatcctgata attaattggc aagaattgag 1350 gcagaagggt gggaaaccaggaccacagcc ccaagtccct tcttatgggt 1400 ggtgggctct tgggccatag ggcacatgccagagaggcca acgactctgg 1450 agaaaccatg agggtggcca tcttcgcaag tggctgctccagtgatgagc 1500 caacttccca gaatctgggc aacaactact ctgatgagcc ctgcatagga1550 caggagtacc agatcatcgc ccagatcaat ggcaactacg cccgcctgct 1600ggacacagtt cctctggatt atgagtttct ggccactgag ggcaaaagtg 1650 tctgttaaaaatgccccatt aggccaggat ctgctgacat aattgcctag 1700 tcagtccttg ccttctgcatggccttcttc cctgctacct ctcttcctgg 1750 atagcccaaa gtgtccgcct accaacactggagccgctgg gagtcactgg 1800 ctttgccctg gaatttgcca gatgcatctc aagtaagccagctgctggat 1850 ttggctctgg gcccttctag tatctctgcc gggggcttct ggtactcctc1900 tctaaatacc agagggaaga tgcccatagc actaggactt ggtcatcatg 1950cctacagaca ctattcaact ttggcatctt gccaccagaa gacccgaggg 2000 aggctcagctctgccagctc agaggaccag ctatatccag gatcatttct 2050 ctttcttcag ggccagacagcttttaattg aaattgttat ttcacaggcc 2100 agggttcagt tctgctcctc cactataagtctaatgttct gactctctcc 2150 tggtgctcaa taaatatcta atcataacag c 2181 12 24DNA artificial sequence Synthetic oligonucleotide probe 12 tcgcggagctgtgttctgtt tccc 24 13 50 DNA artificial sequence Syntheticoligonucleotide probe 13 tgatcgcgat ggggacaaag gcgcaagctc gagaggaaactgttgtgcct 50 14 20 DNA artificial sequence Synthetic oligonucleotideprobe 14 acacctggtt caaagatggg 20 15 24 DNA artificial sequenceSynthetic oligonucleotide probe 15 taggaagagt tgctgaaggc acgg 24 16 20DNA artificial sequence Synthetic oligonucleotide probe 16 ttgccttactcaggtgctac 20 17 20 DNA artificial sequence Synthetic oligonucleotideprobe 17 actcagcagt ggtaggaaag 20 18 24 DNA artificial sequenceSynthetic oligonucleotide probe 18 tatccctcca attgagcacc ctgg 24 19 21DNA artificial sequence Synthetic oligonucleotide probe 19 gtcggaagacatcccaacaa g 21 20 24 DNA artificial sequence Synthetic oligonucleotideprobe 20 cttcacaatg tcgctgtgct gctc 24 21 24 DNA artificial sequenceSynthetic oligonucleotide probe 21 agccaaatcc agcagctggc ttac 24 22 50DNA artificial sequence Synthetic oligonucleotide probe 22 tggatgaccggagccactac acgtgtgaag tcacctggca gactcctgat 50 23 260 PRT Homo sapiens23 Leu Ala Leu Gly Ser Val Thr Val His Ser Ser Glu Pro Glu Val 1 5 10 15Arg Ile Pro Glu Asn Asn Pro Val Lys Leu Ser Cys Ala Tyr Ser 20 25 30 GlyPhe Ser Ser Pro Arg Val Glu Trp Lys Phe Asp Gln Gly Asp 35 40 45 Thr ThrArg Leu Val Cys Tyr Asn Asn Lys Ile Thr Ala Ser Tyr 50 55 60 Glu Asp ArgVal Thr Phe Leu Pro Thr Gly Ile Thr Phe Lys Ser 65 70 75 Val Thr Arg GluAsp Thr Gly Thr Tyr Thr Cys Met Val Ser Glu 80 85 90 Glu Gly Gly Asn SerTyr Gly Glu Val Lys Val Lys Leu Ile Val 95 100 105 Leu Val Pro Pro SerLys Pro Thr Val Asn Ile Pro Ser Ser Ala 110 115 120 Thr Ile Gly Asn ArgAla Val Leu Thr Cys Ser Glu Gln Asp Gly 125 130 135 Ser Pro Pro Ser GluTyr Thr Trp Phe Lys Asp Gly Ile Val Met 140 145 150 Pro Thr Asn Pro LysSer Thr Arg Ala Phe Ser Asn Ser Ser Tyr 155 160 165 Val Leu Asn Pro ThrThr Gly Glu Leu Val Phe Asp Pro Leu Ser 170 175 180 Ala Ser Asp Thr GlyGlu Tyr Ser Cys Glu Ala Arg Asn Gly Tyr 185 190 195 Gly Thr Pro Met ThrSer Asn Ala Val Arg Met Glu Ala Val Glu 200 205 210 Arg Asn Val Gly ValIle Val Ala Ala Val Leu Val Thr Leu Ile 215 220 225 Leu Leu Gly Ile LeuVal Phe Gly Ile Trp Phe Ala Tyr Ser Arg 230 235 240 Gly His Phe Asp ArgThr Lys Lys Gly Thr Ser Ser Lys Lys Val 245 250 255 Ile Tyr Ser Gln Pro260 24 270 PRT Homo sapiens 24 Val Arg Val Thr Val Asp Ala Ile Ser ValGlu Thr Pro Gln Asp 1 5 10 15 Val Leu Arg Ala Ser Gln Gly Lys Ser ValThr Leu Pro Cys Thr 20 25 30 Tyr His Thr Ser Thr Ser Ser Arg Glu Gly LeuIle Gln Trp Asp 35 40 45 Lys Leu Leu Leu Thr His Thr Glu Arg Val Val IleTrp Pro Phe 50 55 60 Ser Asn Lys Asn Tyr Ile His Gly Glu Leu Tyr Lys AsnArg Val 65 70 75 Ser Ile Ser Asn Asn Ala Glu Gln Ser Asp Ala Ser Ile ThrIle 80 85 90 Asp Gln Leu Thr Met Ala Asp Asn Gly Thr Tyr Glu Cys Ser Val95 100 105 Ser Leu Met Ser Asp Leu Glu Gly Asn Thr Lys Ser Arg Val Arg110 115 120 Leu Leu Val Leu Val Pro Pro Ser Lys Pro Glu Cys Gly Ile Glu125 130 135 Gly Glu Thr Ile Ile Gly Asn Asn Ile Gln Leu Thr Cys Gln Ser140 145 150 Lys Glu Gly Ser Pro Thr Pro Gln Tyr Ser Trp Lys Arg Tyr Asn155 160 165 Ile Leu Asn Gln Glu Gln Pro Leu Ala Gln Pro Ala Ser Gly Gln170 175 180 Pro Val Ser Leu Lys Asn Ile Ser Thr Asp Thr Ser Gly Tyr Tyr185 190 195 Ile Cys Thr Ser Ser Asn Glu Glu Gly Thr Gln Phe Cys Asn Ile200 205 210 Thr Val Ala Val Arg Ser Pro Ser Met Asn Val Ala Leu Tyr Val215 220 225 Gly Ile Ala Val Gly Val Val Ala Ala Leu Ile Ile Ile Gly Ile230 235 240 Ile Ile Tyr Cys Cys Cys Cys Arg Gly Lys Asp Asp Asn Thr Glu245 250 255 Asp Lys Glu Asp Ala Arg Pro Asn Arg Glu Ala Tyr Glu Glu Pro260 265 270 25 263 PRT Homo sapiens 25 Leu Cys Ser Leu Ala Leu Gly SerVal Thr Val His Ser Ser Glu 1 5 10 15 Pro Glu Val Arg Ile Pro Glu AsnAsn Pro Val Lys Leu Ser Cys 20 25 30 Ala Tyr Ser Gly Phe Ser Ser Pro ArgVal Glu Trp Lys Phe Asp 35 40 45 Gln Gly Asp Thr Thr Arg Leu Val Cys TyrAsn Asn Lys Ile Thr 50 55 60 Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu ProThr Gly Ile Thr 65 70 75 Phe Lys Ser Val Thr Arg Glu Asp Thr Gly Thr TyrThr Cys Met 80 85 90 Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val LysVal Lys 95 100 105 Leu Ile Val Leu Val Pro Pro Ser Lys Pro Thr Val AsnIle Pro 110 115 120 Ser Ser Ala Thr Ile Gly Asn Arg Ala Val Leu Thr CysSer Glu 125 130 135 Gln Asp Gly Ser Pro Pro Ser Glu Tyr Thr Trp Phe LysAsp Gly 140 145 150 Ile Val Met Pro Thr Asn Pro Lys Ser Thr Arg Ala PheSer Asn 155 160 165 Ser Ser Tyr Val Leu Asn Pro Thr Thr Gly Glu Leu ValPhe Asp 170 175 180 Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr Ser Cys GluAla Arg 185 190 195 Asn Gly Tyr Gly Thr Pro Met Thr Ser Asn Ala Val ArgMet Glu 200 205 210 Ala Val Glu Arg Asn Val Gly Val Ile Val Ala Ala ValLeu Val 215 220 225 Thr Leu Ile Leu Leu Gly Ile Leu Val Phe Gly Ile TrpPhe Ala 230 235 240 Tyr Ser Arg Gly His Phe Asp Arg Thr Lys Lys Gly ThrSer Ser 245 250 255 Lys Lys Val Ile Tyr Ser Gln Pro 260 26 273 PRT Homosapiens 26 Leu Cys Ala Val Arg Val Thr Val Asp Ala Ile Ser Val Glu Thr 15 10 15 Pro Gln Asp Val Leu Arg Ala Ser Gln Gly Lys Ser Val Thr Leu 2025 30 Pro Cys Thr Tyr His Thr Ser Thr Ser Ser Arg Glu Gly Leu Ile 35 4045 Gln Trp Asp Lys Leu Leu Leu Thr His Thr Glu Arg Val Val Ile 50 55 60Trp Pro Phe Ser Asn Lys Asn Tyr Ile His Gly Glu Leu Tyr Lys 65 70 75 AsnArg Val Ser Ile Ser Asn Asn Ala Glu Gln Ser Asp Ala Ser 80 85 90 Ile ThrIle Asp Gln Leu Thr Met Ala Asp Asn Gly Thr Tyr Glu 95 100 105 Cys SerVal Ser Leu Met Ser Asp Leu Glu Gly Asn Thr Lys Ser 110 115 120 Arg ValArg Leu Leu Val Leu Val Pro Pro Ser Lys Pro Glu Cys 125 130 135 Gly IleGlu Gly Glu Thr Ile Ile Gly Asn Asn Ile Gln Leu Thr 140 145 150 Cys GlnSer Lys Glu Gly Ser Pro Thr Pro Gln Tyr Ser Trp Lys 155 160 165 Arg TyrAsn Ile Leu Asn Gln Glu Gln Pro Leu Ala Gln Pro Ala 170 175 180 Ser GlyGln Pro Val Ser Leu Lys Asn Ile Ser Thr Asp Thr Ser 185 190 195 Gly TyrTyr Ile Cys Thr Ser Ser Asn Glu Glu Gly Thr Gln Phe 200 205 210 Cys AsnIle Thr Val Ala Val Arg Ser Pro Ser Met Asn Val Ala 215 220 225 Leu TyrVal Gly Ile Ala Val Gly Val Val Ala Ala Leu Ile Ile 230 235 240 Ile GlyIle Ile Ile Tyr Cys Cys Cys Cys Arg Gly Lys Asp Asp 245 250 255 Asn ThrGlu Asp Lys Glu Asp Ala Arg Pro Asn Arg Glu Ala Tyr 260 265 270 Glu GluPro 27 413 DNA Homo Sapien 27 ctcgagccgc tcgagccgtg cggggaaatatcgttgtgaa gttagtgccc 50 catctgagca aggccaaaac ctggaagagg atacagtcactctggaagta 100 ttagtggctc cagcagttcc atcatgtgaa gtaccctctt ctgctctgag150 tggaactgtg gtagagctac gatgtcaaga caaagaaggg aatccagctc 200ctgaatacac atggtttaag gatggcatcc gtttgctaga aaatcccaga 250 cttggctcccaaagcaccaa cagctcatac acaatgaata caaaaactgg 300 aactctgcaa tttaatactgtttccaaact ggacactgga gaatattcct 350 gtgaagcccg caattctgtt ggatatcgcaggtgtcctgg ggaaacgaat 400 gcaagtagat gat 413 28 22 DNA artificialsequence Synthetic oligonucleotide probe 28 atcgttgtga agttagtgcc cc 2229 23 DNA artificial sequence Synthetic oligonucleotide probe 29acctgcgata tccaacagaa ttg 23 30 48 DNA artificial sequence Syntheticoligonucleotide probe 30 ggaagaggat acagtcactc tggaagtatt agtggctccagcagttcc 48

What is claimed is:
 1. A method of treating an inflammatory disease byadministrating a therapeutically effective amount of a PRO301, PRO362 orPRO245 antagonist, or fragment thereof.
 2. The method of claim 1 whereinthe antagonist is an antibody.
 3. The method of claim 2 wherein theantibody is a monoclonal antibody.
 4. The method of claim 3 wherein theantibody has non-human complementarity deterrnining region (CDR)residues and contains human framework region (FR) residues.
 5. Themethod of claim 4 wherein the antibody is a composition in admixturewith a pharmaceutically acceptable carrier or excipient.
 6. The methodof claim 5 wherein the inflammatory disease is selected from the groupconsisting of: inflammatory bowel disease; systemic lupus erythematosis;rheumatoid arthritis; juvenile chronic arthritis; spondyloarthropathies;systemic sclerosis, for example, scleroderma; idiopathic inflammatorymyopathies for example, dermatomyositis, polymyositis; Sjögren'ssyndrome; systemic vaculitis; sarcoidosis; autoimmune hemolytic anemiafor example, immune pancytopenia, paroxysmal nocturnal hemoglobinuria;autoimmune thrombocytopenia, for example, idiopathic thrombocytopenicpurpura, immune-mediated thrombocytopenia; thyroiditis, for example,Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis; diabetes mellitus, immune-mediatedrenal disease, for example, glomerulonephritis, tubulointerstitialnephritis; demyelinating diseases of the central and peripheral nervoussystems such as multiple sclerosis, idiopathic polyneuropathy;hepatobiliary diseases such as infectious hepatitis such as hepatitis A,B, C, D, E and other nonhepatotropic viruses; autoimmune chronic activehepatitis; primary biliary cirrhosis; granulomatous hepatitis; andsclerosing cholangitis; inflammatory and fibrotic lung diseases (e.g.,cystic fibrosis); gluten-sensitive enteropathy; Whipple's disease;autoimmune or immune-mediated skin diseases including bullous skindiseases, erythema multiforme and contact dermatitis, psoriasis;allergic diseases of the lung such as eosinophilic pneumonias,idiopathic pulmonary fibrosis and hypersensitivity pneumonitis,transplantation associated diseases including graft rejection andgraft-verus host disease.
 7. A method for determining the presence of aPRO301, PRO362 or PRO245 polypeptide comprising exposing a cellsuspected of containing the polypeptide to an anti-PRO301, anti-PRO362or anti-245 antibody and determining the binding of the antibody to thecell.
 8. A method of diagnosing an inflammatory disease in a mammal,comprising detecting the level of expression of a gene encoding aPRO301, PRO362 or PRO245 (a) in a test sample of tissue cells obtainedfrom the mammal, and (b) in a control sample of known normal tissuecells of the same cell type, wherein a higher expression level in thetest sample indicated the presence of an inflammatory disease in themammal.
 9. A method of diagnosing an inflammatory disease in a mammal,comprising: (a) contacting an anti-PRO301, anti-PRO362 or anti-PRO245antibody with a test sample of tissue culture cells obtained from themammal, and (b) detecting the formation of a complex between theantibody and the PRO301, PRO362 or PRO245 polypeptide.
 10. A method offor inhibiting the growth of tumor cells comprising exposing a cellwhich overexpresses a PRO30 1, PRO362 or PRO245 polypeptide to aneffective amount of an agent inhibiting the expression and/or activityof the PRO301, PRO362 or PRO245 polypeptide, respectively.
 11. Themethod of claim 10 wherein said agent is an anti-PRO301, anti-PRO362 oranti-PRO245 antibody.
 12. The method of claim 11, wherein said tumorcells are further exposed to radiation treatment or a cytotoxic orchemotherapeutic agent.
 13. A method diagnosing tumor in a mammal,comprising detecting the level of expression of a gene encoding aPRO301, PRO362 or PRO245 polypeptide (a) in a test sample of tissuecells obtained from the mammal, and (b) in a control sample of knownnormal tissue cells of the same cell type, wherein a higher expressionlevel in the test sample indicates the presence of tumor in the mammalfrom the test tissue cells were obtained.
 14. A method of diagnosingtumor in a mammal, comprising (a) contacting an anti-PRO301, anti-PRO362or anti-PRO245 antibody with a test sample of tissue cells obtained fromthe mammal, and (b) detecting the formation of a complex between theanti-PRO301, anti-PRO362 or anti-PRO245 antibody and the PRO301, PRO362or PRO245, respectively, in the test sample.
 15. The method of claim 14wherein said test sample is obtained from an individual suspected tohave neoplastic cell growth or proliferation.
 16. An isolated antibodywhich binds a PRO301 or PRO362 polypeptide.
 17. The antibody of claim 16which is a monoclonal antibody.
 18. The antibody of claim 17 whichcontains non-human complementarity determining region (CDR) residues andhuman framework region (FR) residues.
 19. The antibody of claim 18 whichis labeled.
 20. The antibody of claim 19 which is immobilized on a solidsupport
 21. The antibody of claim 16 which is an antibody fragment, asingle-chain antibody, or an anti-idiotypic antibody.
 22. A compositioncomprising the antibody of claim 21 in admixture with apharmaceutically-acceptable carrier.
 23. The composition of claim 22further comprising a second antibody or a cytotoxic or chemotherapeuticagent.
 24. Isolated nucleic acid comprising DNA having at least a 95%sequence identity to (a) a DNA molecule encoding a PRO301 polypeptidecomprising the sequence of amino acids 28 to 235 of FIG. 2 (SEQ ID NO:1).
 25. The isolated nucleic acid of claim 24 comprising DNA having atleast a 95% sequence identity to (a) a DNA molecule encoding a PRO301polypeptide comprising the sequence of amino acids 28 to 258 of FIG. 2(SEQ ID NO: 1 ), or (b) the complement of the DNA molecule of (a). 26.The isolated nucleic acid of claim 25 comprising DNA having at least a95% sequence identity to (a) a DNA molecule encoding a PRO301polypeptide comprising the sequence of amino acids 28 to 299 of FIG. 2(SEQ ID NO: 1), or (b) the complement of the DNA molecule of (a). 27.The isolated nucleic acid of claim 26 comprising DNA having at least a95% sequence identity to (a) a DNA molecule encoding a PRO301polypeptide comprising the sequence of amino acids 1 to 299 of FIG. 2(SEQ ID NO: 1), or (b) the complement of the DNA molecule of (a). 28.The isolated nucleic acid of claim 24 comprising DNA encoding a PRO301polypeptide having amino acid residues 28 to 235 of FIG. 2 (SEQ ID NO:1).
 29. The isolated nucleic acid of claim 28 comprising DNA encoding aPRO301 polypeptide having amino acid residues 28 to about 258 of FIG. 2(SEQ ID NO: 1).
 30. The isolated nucleic acid of claim 29 comprising DNAencoding a PRO301 polypeptide having amino acid residues 28 to 299 ofFIG. 2 (SEQ ID NO: 1).
 31. The isolated nucleic acid of claim 30comprising DNA encoding a PRO301 polypeptide having amino acid residues1 to 299 of FIG. 2 (SEQ ID NO: 1).
 32. Isolated nucleic acid comprisingDNA having at least 80% sequence identity to (a) a DNA molecule encodinga PRO362 polypeptide comprising the sequence of amino acid residues 1 to321 of FIG. 3 (SEQ ID NO: 2), or (b) the complement of the DNA moleculeof (a).
 33. The nucleic acid of claim 32, wherein said DNA comprises thenucleotide sequence of SEQ ID NO: 8 or its complement.
 34. The nucleicacid of claim 32, wherein said DNA comprises nucleotides 119-1081 of thenucleotide sequence of SEQ ID NO:
 7. 35. An isolated nucleic acidcomprising DNA having at least a 95% sequence identity to (a) a DNAmolecule encoding the same mature polypeptide encoded by the cDNA inATCC Deposit No. 209432 (designation: DNA40628-1216), or (b) thecomplement of the DNA molecule of (a).
 36. The isolated nucleic acid ofclaim 35 comprising then PRO301 encoding sequence of the cDNA in ATCCdeposit No. (designation: DNA40628-1216), or a sequence which hybridizesthereto under stringent conditions.
 37. Isolated nucleic acid comprisingDNA having at least an 80% sequence identity to (a) a DNA moleculeencoding the same mature polypeptide encoded by the human protein cDNAin ATCC Deposit No.: 209620 (DNA45416-1251), or (b) the complement ofthe DNA molecule of (a).
 38. The nucleic acid of claim 37 whichcomprises a DNA molecule encoding the same mature polypeptide encoded bythe human protein cDNA in ATCC Deposit No.: 209620 (DNA45416-1251). 39.Isolated nucleic acid comprising DNA having at least an 80% sequenceidentity to (a) a DNA molecule encoding a PRO362 polypeptide comprisingthe sequence of amino acid residues 1 to X of FIG. 3 (SEQ ID NO: 2), or(b) the complement of the DNA molecule of (a), wherein X is any one ofamino acid residues 271 to 280 of FIG. 3 (SEQ ID NO: 2).
 40. A processfor producing PRO301, PRO362 or PRO245 polypeptides comprising culturinga host cell under conditions suitable for expression of PRO301, PRO362or PRO245 and recovering PRO301, PRO362 or PRO245, respectively, fromthe cell culture.
 41. Isolated native sequence PRO301 polypeptidecomprising amino acid residues 28 to 235 of FIG. 2 (SEQ ID NO: 1). 42.The isolated native sequence PRO301 polypeptide of claim 41 furthercomprising amino acid residues 28 to about 258 of FIG. 2 (SEQ ID NO: 1).43. The isolated native sequence PRO301 polypeptide of claim 42 furthercomprising amino acid residues 28 to 299 of FIG. 2 (SEQ ID NO: 1). 44.The isolated native sequence PRO301 polypeptide of claim 43 furthercomprising amino acid residues 1 to 299 of FIG. 2 (SEQ ID NO: 1). 45.Isolated native sequence PRO301 polypeptide encoded by the nucleic aciddeposited under accession number ATCC
 209432. 46. Isolated nativesequence PRO362 polypeptide comprising amino acid residues 1 to 229 ofFIG. 3 (SEQ ID NO: 2).
 47. Isolated PRO362 polypeptide -comprising aminoacids 1 to X of the amino acid sequence shown in FIG. 3 (SEQ ID NO: 2),wherein X is any one or amino acids 271 to
 280. 48. Isolated PRO362polypeptide encoded by the cDNA insert of the vector deposited as ATCCAccession No. 209432 (DNA45416-1251).